Download - Activation of mechanonociceptors by pro-inflammatory peptides melittin and PLAP peptide

Exp Brain Res (1994) 100:18-28 �9 Springer-Verlag 1994

Brian Cooper - John S. Bomalaski

Activation of mechanonociceptors by pro-inflammatory peptides melittin and PLAP peptide

Received: 5 May 1993 / Accepted: 25 February 1994

Abstract Studies were conducted to characterize the chemical reactivity of A and C fiber mechanical and mechanoheat nociceptors that innervate the goat palatal mucosa. In mechanical nociceptors, no significant chemical reactivity to either serotonin, bradykinin, prostaglandin El, or prostaglandin E2 was observed, regardless of whether substances were injected singly or in spaced sequential combinations. Weak reactivity was observed in mechanoheat nociceptors. In contrast, both mechanical and mechanoheat nociceptors were activated by novel proinflammatory peptides. Nineteen of 30 mechanonociceptors and 12 of 13 mechanoheat nociceptors were activated by the insect venom peptide, melittin or its endogenous mammalian homologue PLAP (phospholipase A2 activating protein) peptide. Low threshold mechanoreceptors were never activated by melittin or PLAP peptide.

Key words Nociceptors �9 Serotonin �9 Bradykinin PLAP �9 Goat

Introduction

Although the thermal reactivity of nociceptors has been relatively well characterized (see Campbell et al. 1989), their chemical and mechanical response properties are less clear. The sensitivity of various nociceptor groups to endogenous chemical stimuli is clearly important. The direct activation of nociceptors by hormones, auto- coids and other tissue constituents is likely to make a contribution to acute and chronic inflammatory pain

B. Cooper (l~) Departments of Oral and Maxillofacial Surgery, Oral Biology and Neuroscience, JHM Health Center, University of Florida, Gainesville, Florida 32610, USA, FAX no: (904)-392-7609 J. S. Bomalaski Department of Medicine, Medical College of Pennsylvania, 3300 Henry Ave, Philadelphia, PA 19129, USA

via the direct sensory consequences of afferent activity and the effect this activity has on central convergent relays (central sensitization; Meller and Gebhart 1993). Potentially, both myelinated and unmyelinated pools of afferents are activated by endogenous pain-producing substances.

Studies from a variety of preparations suggest that unmyelinated nociceptors possess a substantially greater capacity for chemical activation than their myelinated counterparts. Substances have been identified which activate a considerable portion of this group of afferents, while others apparently activate only a subset. In preparations utilizing rabbit, rat, cat, monkey and dog, it has frequently been reported that bradykinin and/or serotonin can activate a large portion of the heat sensitive, unmyelinated superficial and deep nociceptor pool. De- pending on the preparation and method of administration, 60% of the characterized, unmyelinated afferents respond to one or both of these substances (e.g., Herbert and Schmidt 1992; Khan et al. 1992; Mizumura et al. 1992; Sengupta et al. 1992; but see Chahl and Iggo 1977). Other agents, such as histamine, acetylcholine, and protons also activate unmyelinated nociceptors, but their effectiveness is confined to about 40% of nociceptors (Heppelmann et al. 1987; Kumazawa and Mizu- mura 1977, 1980; Lang et al. 1990; Steen et al. 1992). Eicosanoids, such as leukotriene B 4 (LTB4), prostaglandins Ea and El, and 8,15 dihydroxyeicosatetranoic acid (8,15 diHete), which play prominent roles in en- hancing reactivity of afferents to thermal, mechanical, and sometimes chemical stimuli are rarely reported to activate these afferents directly (Birrell et al. 1991; Hep- plemann et al. 1987; Madison et al. 1992).

Chemical stimulation of myelinated nociceptors has received relatively less attention. In contrast to C mechanoheat/polymodal nociceptors, A delta afferent responses to bradykinin or serotonin are relatively poor or non-existent in cutaneous preparations (Burgess and Perl 1967; Khan et al. 1992; Martin et al. 1987; Perl 1968; Szolcsanyi 1987; Beck and Handwerker 1974; but see Fjallbrant and Iggo 1961), but may be weakly re-

sponsive in deep afferent pools (He et al. 1990; Hepple- mann et al.. 1987; Herbert and Schmidt 1992; Kuma- zawa and Mizumura 1977, 1980; Mense 1982; Schaible and Schmidt 1988; Sengupta et al. 1992).

Although studies have focussed on the algesic properties of bradykinin, serotonin, and prostaglandins, it is likely that other mediators will be identified that possess the capacity to activate or modulate the activation of nociceptors. Inflammation is a complex event involving, hormonal, paracrine, and autocrine mediators that dre derived from diverse sources (Cooper and Sessle 1992; Gallin et al. 1992). These substances contribute to pain in a variety of ways, including sensitization and activation of nociceptors and the production of edema.

One novel substance is the recently identified and sequenced protein, PLAP (phospholipase A2 activating protein; Bomalaski and Clark 1993). Human PLAP was first isolated from the synovial fluid of patients with rheumatoid arthritis as that substance recognized by antibodies raised against the bee venom peptide, melittin (Bomalaski et al. 1989; Clark et al. 1987; Clark et al. 1988). Subsequent sequencing of the PLAP protein revealed regions of melittin homology (Clark et al. 1991). Both PLAP and synthetic peptide fragments of PLAP containing the melittin homology were determined to have functional capacities similar to melittin (Boma- laski and Clark 1993).

The central role of PLA2 (phospholipase A2) in inflammatory events is well known (Bomalaski and Clark 1993). PLAP may play a pivotal role in the development and/or maintenance of inflammation by mediating, the activation of PLA 2 by autocrine and paracrine signals. The crucial role of PLAP in the activation of PLA2, its presence in the synovial fluid of patients with inflammatory arthritis and its mimicry by a pro-inflammatory insect venom suggested that it was an interesting candi- date as an activator of nociceptors. We decided to test this substance in the goat palatal preparation.

The goat palatal mucosa contains a rich innervation of myelinated and unmyelinated nociceptors (Halata and Cooper 1991). The palate - by virtue of its direct apposition to the maxillary bone - offers excellent conditions for the delivery of controlled, intense, mechanical stimuli. Studies in the goat palatal preparation have revealed specialized sub-populations of high threshold mechanoreceptors (mechanonociceptors, intense pressure receptors). These afferents have conduction velocities primarily in the A delta range, but some may be found in the A beta range as well. They are clearly dis- tinguished from low threshold mechanoreceptors by virtue of their thresholds (2-310 g) and substantial response range. Mechanonociceptors (MN) are an espe- cially interesting subgroup of high threshold mechanoreceptors. They have very high thresholds (75-310 g), transduce stimuli in a range consistent with human pain report (Cooper et al. 1993), possess an extensive capacity for sensitization when exposed to pro-inflammatory mediators (Cooper and Friedman 1994; Cooper et al. 1991b), and are sensitive to changes in the mechanical

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transmission properties of tissues consequent to edema (Cooper 1993). We have yet to examine the chemical response repertoire of these nociceptors.

In the present report, we examined the chemical reactivity of palatal nociceptors to novel peptides and to substances (serotonin, bradykinin, prostaglandin El, and prostaglandin E2) which have been demonstrated to activate portions of the myelinated and unmyelinated nociceptor pool. Despite single and sequential injections, no substantial activation of MNs could be demonstrated to classic mediators, but - as is typically reported for cutaneous preparations - a portion of the A delta and C mechanoheat nociceptors were activated by bradykinin or serotonin. In contrast, both mechanonociceptors and mechanoheat nociceptors were activated by the insect venom peptide melittin and its anti- genic and functional homologue, PLAP peptide. A portion of this data has been previously published in abstract form (Cooper and Bomalaski 1993).

Materials and methods Subjects

Seventy-three male goats (Capra hireus) served as subjects. Goats were housed on pasture or in runs maintained by the University of Florida Veterinary Hospital. Health and care of the goats was supervised by the University of Florida veterinary and animal care staff. All procedures employed in the present study were reviewed and approved by the local committee on animal care.

Exposure of and recording from the trigeminal ganglion

Subjects were anesthetized with a combination of alpha-chloralose and sodium pentobarbital (i.v.). Chloralose was administered via a Ringer's drip (7.5 mg/ml). Both substances were administered to effect. After an extensive dissection of the vessels and glands caudal to the mandibular ramus, the trigeminal ganglion was exposed by removal of a small piece of temporal bone just caudal to the oval foramen. The ganglion was penetrated with either glass pipettes or tungsten microelectrodes (Micro Probe Inc.) until receptive fields were located on the palate (Fig. 1). Com- plete descriptions of surgical dissection and recording procedures have been previously published (Cooper et al. 1991a).

Materials and procedures

A 21 amino acid peptide fragment of the PLA2 activating protein (PLAP peptide) was obtained from the laboratory of J.S. Boma- laski or was synthesized at the protein core facility of the Depart- ment of Biochemistry of the University of Florida according to sequences identified and characterized by Dr. Bomalaski and col- leagues (Clark et al. 1991). Other substances were obtained com- mercially. Serotonin, prostaglandins E1 and E2, bradykinin (Sig- ma), melittin (Sigma and Research Biochemicals Inc.) and PLAP peptide were dissolved in physiological saline (37~ prior to injection.

Receptive felds were identified and preliminary characteriza- tions were conducted with Von Frey hairs (monofilament nylon type). Von Frey hairs were flame rounded to prevent confounding of application pressures by jagged edges (see Cooper et al. 1993). Subsequent characterization was made with a servo controlled mechanical stimulator.

Injections were made in the proximity of the receptive field using microsyringes (5 or 10 gl capacity; Fisher Chemical). Agents

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Fig. 1 The incisal papilla (IP) of the goat. The IP of the goat is a specialized portion of the maxillary palatal mucosa. The IP is viewed from the side in the upper panel and from below in the middle panel. Labels refers to subdivisions of the palate (I/Z ven- tral zone, MZ medial zone, SZ sulcal zone, LBZ lateral border zone). The lower frame shows the relationship of the IP to other oral cavity structures

were injected singly PGE 1 (n=13); PGE 2 (n= l l ) ; serotonin (n=16); bradykinin (n=15); melittin (n=19); PLAP peptide (n = 1) or in sequences. Of the many injection sequences used, the following were most common: serotonin-PGE2 (n = 5); serotonin- PGE~ (n=9); PGE2-serotonin (n=6); PGEl-serotonin (n=5); PGEj or PGE2-bradykinin (n=5); serotonin-PGE~-melittin (n=6); bradykinin-melittin (n=8); bradykinin-PLAP peptide (n=5); PGE2-serotonin-bradykinin-PLAP peptide (n=3). Other sequences are described in Table 2. Dosage levels were administered with quantities as salts when indicated: bradykinin acetate (5 gg or 100 gg; 4.5 and 90 nmol); serotonin hydrochloride (12 gg; 56 nmol)~ PGE 1 or PGE 2 (5 gg; 14 nmol each); melittin (5, 8 or 16 gg; 1.75, 2.8, or 5.6 nmol); PLAP peptide (50 Ixg, 125 gg or 250 I.tg; 15, 37 or 75 nmol). Doses of bradykinin, serotonin, and

prostaglandins that were used were based on those commonly employed in electrophysiological studies 0.5-30 gg bradykinin; 1-150 i.tg serotonin; 0.5 to 30 gg PGE; see Grubh et al. 1991; Mense 1981; Schaible and Schmidt 1988). Melittin doses were initially matched to bradykinin (weight) and graded up from there. PLAP peptide doses were initially matched to melittin and increased to a level where they became effective. Administrations were made in 5 gl (or rarely 10 gl) volumes. The 90 nmol dosage of bradykinin was used in only two instances.

Needles penetrated the papilla at a distance of 10-15 mm from the receptive field. The needle was brought into the vicinity of the receptive field (within 5 mm) and the substance was injected over a period of 1-2 s. With some exceptions, at least 30 min passed between consecutive injections of substances. Injections of saline vehicle were often made prior to, or as part of a sequence of injections (n = 28).

Afferent activity was digitized and stored on a Vetter 3000 and Panasonic VHS unit (Pacer Electronics). To examine gross reactivity to injected substances, activity was printed continuously, online, for several minutes prior to and following injection on a thermal printer (DASH IV, Astromed; see Fig. 2). In order to determine the identity of the activity evoked as well as the peak rate and mean rate of this activity, stored data were printed in high resolution (10 kHz sampling rate), 10 s blocks by recapturing stored data on the 256K memory card of the DASH IV.

Statistics

Detailed activity records were divided into 1000 ms bins. The mean rate of chemical activation was the mean of activity observed in a 1000 ms bin during a peak activation period. Peak activation periods were identified by inspection of on-line print outs of activity. Actual rate calculations were made from detailed records that were recaptured from VHS storage tapes. Peak periods were usually 10-40 s after the injection of a substance.

In order to evaluate the degree of chemical activation, a method of "equivalent intensities" was used. Equivalent intensities were determined by comparing the peak rate during chemical stimulation to the rate observed during mechanical stimulation. The peak rate was calculated as the highest rate achieved by seven impulses (six intervals) during the peak activation period. Six intervals were chosen, as this approximated the method used to construct mechanical transduction functions (Cooper et al. 1991a). In this manner, the degree of activation of the afferent by the algesic was evaluated in terms of equivalent pressures required to produce a relatively equal rate of activation over the same time period.

Conduction time was determined by electrical stimulation of the receptive field. The time between the electrical artifact and the arrival of the action potential at the recording electrode was determined by inspection of printed records of the event. Conduction distance was divided by the conduction time to arrive at the conduction velocity. C fibers were considered to be those afferents with conduction velocities less than 2.5 m/s. A beta fibers were considered to be those with conduction velocities greater than 30 m/s. Fibers were considered to be A delta when the conduction velocity fell between these two values.

Transduction by nociceptors

Most afferents were characterized with respect to their capacity to transduce intense mechanical stimuli. Ramped or stepped stimulus presentations of force were made in either displacement or force-servo mode using a 2 mm diameter, spherically tipped probe mounted on an HC stimulator (a force/displacement servo stimulator developed by B.C. and William Hargens, Professional Engi- neer; Cooper and Hargens 1993). The instantaneous relationship between force and interval formed data pairs that were pooled and fit to power functions using simple linear regression (SAS). Separate regression lines were determined for dynamic and static

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Table 1 Peak and mean rate of activity of A and C mechanoheat nociceptors following injection of bradykinin or serotonin. Com- pounds were injected near the receptive field of the afferent. Reac- tivity to bradykinin is indicated by the presence of a peak and mean rate score in the upper portion of the table. Reactivity to serotonin is indicated by the presence of a peak and mean rate score in the lower portion of the table. Transduction range of each afferent is given as the pressure over which a significant relation-

ship could be described between instantaneous pressure and response interval (1 N/mm2= 100 g/ram2= t0 bars). Equivalent intensity indicates how the activation produced by bradykinin or serotonin would equate to mechanical stimulation. Mechanonoci- ceptors never responded to bradykinin regardless of whether they were pretreated with serotonin or prostaglandin. (5 Serotonin, B bradykinin, S saline, M melittin, N negligible response, NTno test made

Case Conduction Test Peak velocity sequences activity (m/s) (Hz)

Mean Transduction Equivalent activity range intensity (Hz) (N/ram 2) (N/mm 2)

Bradykinin 1 19.30 S, B - - NT - 2 19.80 B 1.8 1.8 0.31~).87 N 3 50.00 B, M, B - - NT - 4 18.10 M, P2, M, B - - 0.17-0.25 - 5" 14.15 S, B 18.8 16.2 0.07-1.34 0.61 6 6.60 B -- - NT --

Serotonin 1 50.00 B, M, B, 5 3.5 3.5 0.06~0.50 N 2 19.80 B, M, B, 5 56.5 54.3 0.31-0.87 0.08 3 14.15 S, B, M, B, 5 8.2 7.7 0.17-1.34 0.15 4 19.30 S, B, M, B, 5 - - NT -

a Dose 90 nmol

reactivity. An F test was used to determine the significance of the fit for each case, and only significant lines were accepted (P < 0.05). In cases in which the receptive fields were difficult to approach with the HC stimulator, the force probe was detached from the translation stage and stimuli were applied by hand. Transduction functions (dynamic stimulus) were used to compute equivalent intensities. A more complete description of the stimulation and quantification procedures is given in previous reports (Cooper et al. 1991a; Cooper and Hargens 1993).

A heat gun (Newark Electronics) was used to assess thermal reactivity. Convection-assisted radiant heat was directed from a fixed distance to the tissue surface for 10 s. This was calibrated to be 53~ by a thermistor.

Results

Chemical and mechanical response profiles were determined for 64, mechanical nociceptors (c.v. = 3.8-47.5 m/ s), and ten mechanohea t nociceptors (c.v. = 4.9-50.0 m/ s) with conduct ion velocities in the A delta and A beta range; three mechanohea t nociceptors (c .v .=0 .4- 1.06 m/s) with conduct ion velocities in the C fiber range; and 17 A beta low threshold mechanoreceptors (LTMs, 31.6-63.3 m/s). A delta and A beta afferents were classified as nociceptors based upon their t ransduct ion capacities (threshold and reactivity range) and conduct ion velocity. Mechanical nociceptors were further sub- grouped as intense pressure receptors (IPR, n = 8) and mechanonociceptors (MN, n = 3 0 ) according to the nomencla ture originated by Perl (i.e., modera te pressure receptors and insensitive mechanoreceptors ; Burgess and Perl 1967; Perl 1968) and subsequently modified by the au thor (Cooper et al. 1991a). Both t ransduced stimuli in ranges that were repor ted to be painful by humans (Cooper et al. 1993). Intense pressure receptors and mechanonociceptors were defined by threshold (Von

Frey) ranges of 2-15 and 15-300 g (20-150 m N and 150-3000 mN) respectively. Mechanonociceptors pref- erentially t ransduced dynamic components of stimuli while intense pressure receptors t ransduced bo th the static as well as the dynamic componen t s (Cooper et al. 1993). By t ransduct ion of dynamic/s ta t ic components , we refer to the capacity to encode by frequency the intensity of a stimulus as it is applied during a constant velocity r amp (dynamic transduction) or a series of s tepped force presentat ions (static transduction). Mechanohea t nociceptors (MH) had propert ies similar to other mechanical nociceptors and, in addition, were heat reactive. A beta L T M s had low thresholds ( < 150- 500 mg), restricted response range, and rapid conduc- t ion velocities. In the c o m m o n terminology, seven LTMs were quickly adapt ing and ten were slowly adapting. Thresholds below 150 mg were not tested.

Effects of serotonin, bradykinin and prostaglandins on mechano- and mechanohea t nociceptors

At least 5 min following mechanical characterization, 5 gl injections of agents were made near the receptive field. With the exception of one or two spikes in several cases, there was no evidence that serotonin (n= 16), prostaglandins (n = 13) or bradykinin (n = 17) could produce any act ivat ion of MNs. In one case, a 90 nmol dose of bradykinin was administered into the receptive field of a mechanonociceptor , but no activity was observed. In contrast, mechanohea t nociceptors were act ivated by bradykinin (two of six cases) or serotonin (three of four cases). Act ivat ion was weak, and generally did not ap- p roach rates observed with mechanical s t imulation of the same afferent (Table 1).

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Fig. 2A-F Activation of an A delta mechanonociceptor by melittin. Melittin produced intense activation of this nociceptor. Following a delay of 9 s, powerful bursting occurred that progressively weakened over a 6 rain ob- servation period (A-F). The post-injection histogram illustrates the distribution of response frequencies in time (1 s bins for 6 rain). In G, the initial intense burst is illustrated as it appeared on a polygraph. Injection is indicated by the a r r o w . Pre-injection "activity" is due to penetration of the tissue with the injection needle. An enlargement of the potential is shown in (H)

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Activation of nociceptors by melittin

Activation of mechanonociceptors (nine of 14) and mechanoheat noeiceptors (eight of nine cases) was observed following the injection of melittin (see Fig. 2, Table 2). Intense pressure receptors (n = 8) and LTMs (n = 9) were never activated. Saline vehicle was always ineffective (n= 18). Pre-exposure to serotonin, PGEs or bradykinin did not potentiate the melittin response (see Table 2). However, ideal conditions for such a test (e.g., the sequence melittin, PGE, melittin) were impaired due to a substantial tachyphylaxis to melittin. Comparisons between different afferents (pre-treated or not) were too variable for an accurate assessment of potentiation.

Activation by melittin was usually slightly delayed. As is typically reported, 10~40 s would pass before activation began (e.g., Herbert and Schmidt 1992; Meyer e t al. 1991; Whalley et al.. 1987), but "instantaneous" responses were also observed on occasion. These delays may have been due to diffusion of the substance from the injection to the receptor site (a distance of 5 mm or less). The presence of delays, the small volume of the injection, and the durat ion of activation made it very unlikely that there was any mechanical component con- tributing to the activation of these afferents.

Melittin activation was intense and prolonged. Typi- cally, irregular bursting discharges continued for many minutes (see Fig. 2). The most intense bursting took

Table 2 Peak and mean rate of activity of A delta and C fiber afferents following injection of melittin. A sequence of compounds was injected near the receptive field of the afferent. Reactivity to melittin is indicated by the presence of a peak and mean rate score. Transduction range of each afferent is given as the pressure over which a significant relationship could be described between

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instantaneous pressure and response interval (1 N/mm2= 100 g/ mm2= 10 bars). Equivalent intensity indicates how the activation produced by melittin would equate to mechanical stimulation. P1 Prostaglandin El, P2 prostaglandin E2, 5 serotonin, B bradykinin, S saline, M melittin, N negligible response, N T n o test made


Mean Transduction Equivalent activity range intensity (Hz) (N/ram 2) (N/mm 2)

MN 1 36.10 M 46.0 2 25.50 M - 3 7.30 M 43.0 4 43.60 M 3.9 5 29.90 M 24.7 6 26.70 5, P1, M 105.3 7 9.40 P1, 5, S, M 27.9 8 6.70 5, P1, S, M 11.8 9 24.90 B, P1, 5, PA, S, M -

10 10.90 P2, 5, M - 11 26.50 P1, 5, S, M 11.0 12 20.70 5, P1, S, M - 13 28.20 B, M 37.3 14 18.10 S, B, M -

MH 1 18.10 M -- 2 19.30 S, B, M 56.2 3 50.00 B, M 17.2 4 19.80 B, M 28.9 5 14.15 B, M 44.8 6 6.60 B, M 18.1 7 1.50 B, M 5.8 8 1.11 M, S 23.8 9 2.54 S, M 17.8

29.5 NT - - 4.17-4.48 -

18.0 0.43-1.66 4.29 3.9 0.70-4.27 1.49

17.6 0.48-2.08 1.38 59.9 1.07-2.43 6.20 25.4 1.02-3.41 3.68 10.0 1.17-3.84 1.95

- 0.25-0.60 - - 3.33-5.99 - 9.0 0.27-2.16 0.41 - 2.08-3.56 -

22.0 0.07-1.12 0.37 - 1.29-1.51 -

- 0.17~0.25 _ 34.4 NT - 11.6 0.06-0.50 0.09 22.8 0.31~).87 0.02 31.8 0.07-1.34 2.65 18.2 NT - 5.4 NT --

20.6 0.10-1.25 0.21 13.6 0.07-0.66 0.28

place soon after the injection; thereafter, there was a general decline th roughou t the post-injection period. Interspersed between bursts were quiet periods which were interrupted by occasional singlet pulses. The discharge rate during peak periods of act ivat ion was 34.4 Hz (SD =28.6; average rate of 21.7, S D = 15.6 Hz) and 33 .0Hz (SD=15 .3 ; average rate of 23.8, S D = 8.5 Hz) for mechanonociceptors and mechanohea t nociceptors, respectively.

An equivalent act ivat ion intensity was determined for each case by compar ing the rate of activation produced by melittin with the t ransduct ion functions formed during initial character izat ion of the afferents (see Table 2). Melittin produced an average peak equivalent intensity of 1.66 N / m m 2 (SD = 1.85; 1660 mN/mm2; 166 g/mm2; 16.6 bars; 664 g of force). This compared to an average t ransduct ion range of 0.90 ( S D = 1.12) to 2.15 (SD = 1.62) N / m m 2 for all mechanonociceptors and mechanohea t nociceptors tested (see Table 2). There- fore, melitt in act ivated nociceptors at rates that corre- sponded to intense stimulation. Typically, this activity fell into a midrange of rates that were characteristic of each afferent. Peak activity induced by melittin actually exceeded observed, mechanical ly induced, maximal rates in four cases (see Table 2, cases 3, 6, and 7, mechanonocicep tor subcategory; also case 5, M H sub-

category) and fell below the min imum mechanical ly induced rate in only one case (Table 2, case 4, M H subcategory).

Activat ion of nociceptors by P L A P peptide

A 21 amino acid peptide fragment of P L A P with a high sequence homology to melittin and powerful PLA2 activating propert ies (Bomalaski and Clark 1993; Clark et al. 1991) was examined for its capacity to activate nociceptors. Following injection of 37-75 nmol, P L A P peptide induced activity in nociceptors that was similar in form, consistency and specificity to its functional homologue, melitt in (see Fig. 3). While doses of 17 nmol were ineffective, higher doses induced burst ing discharges in ten of 16 M N s and four of four M H nociceptors. A beta L T M s (n = 8, five SA and three QA) were never activated by P L A P peptide.

P L A P peptide was not as potent as melittin. Al- though the peak rates of nociceptor activity induced by P L A P peptide were considerable (10.45 Hz, SD = 7.54, for M N s and 9.9 Hz, SD -- 3.2 for M H nociceptors) they were consistently less intense than that produced by melittin. Using the me thod of equivalent intensity, activat ion of M N and M H nociceptors tended to be at or

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12

10

8

6

4

2

Fig. 3 Activation of an A delta mechanonociceptor by PLAP peptide. PLAP peptide produced intense activation of this A delta mechanonociceptor. In the upper panel a polygraph tracing illustrates the reaction of a mechanonociceptor to a 75 nmol injection of PLAP peptide. A single intense burst of activity was observed. In the lower panel the response to PLAP is recon- structed in histogram form. A peak rate of 10 Hz was achieved 2 s after the onset of activity (62 s post injection). The duration of PLAP induced activity was approxi- mately 9 s

RESPONSE TO PLAP PEPTIDE

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Response to PLAP Peptide

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70 80 90 100

slightly above threshold rates achieved during intense mechanical stimulation. On average, this pool of MNs were reactive through a range of 0.50-1.58 N/mm 2 (SD = 0.28 and 0.62, respectively). The mean rate of peak discharge following PLAP peptide equated to 0.69 N/ mm 2 of mechanical stimulation (see Table 3). This fell short of the midrange values typically observed following injection of melittin. In addition, considerably higher doses of PLAP peptide were required to achieve this degree of activation (37-75 nmol vs 1.75 nmol). In two cases (case 6 and 9, Table 3), only ten to 20 pulses were induced by PLAP peptide. Although these were considered to be negligible responses, they were includ- ed in computation of average discharge rate.

Discussion

The effects of bradykinin, serotonin, and prostaglandins on mechano- and mechanoheat nociceptors

any substantial activation of MNs in non-inflamed tissues. However, some activation of MH nociceptors was observed. These findings are consistent with infrequent or no activation of cutaneous A delta afferents in rat, monkey, and rabbit (Khan et al. 1992; Lang et al. 1990; Martin et al. 1987; Szolcsanyi 1987; see also Herbert and Schmidt 1992 for "fast" A delta), but differs from observations in deep structures of cat or dog (joint, muscle: He et al. 1990; Hepplemann et al.. 1987; Herbert and Schmidt 1992; Kanaka et al. 1985; Kumazawa and Mizumura 1977, 1980; Schaible and Schmidt 1988) where weak activation in about half of the afferent pool has been described. It is possible that deep afferents have a broader chemical response profile, or that these afferents are functionally more closely related to the cutaneous MH group. Deep afferents are not typically tested for heat reactivity, and therefore are not usually classified in this way.

The effects of melittin and PLAP peptide

Following extensive testing, we were unable to demon- strate that single or sequential administration of bradykinin, serotonin, PGE 1 or PGE2 could produce

Both PLAP peptide and melittin activated a high pro- portion of A mechano-, mechanoheat, and C mechanoheat nociceptors. Other classes of afferents considered

Table 3 Peak and mean rate of activity of A delta afferents following injection of PLAP. Reactivity to PLAP peptide is indicated by the presence of peak and mean rate scores. The transduction range of each afferent is given as the pressure over which a significant relationship could be described between instantaneous pressure and response interval (1 N/mm2= 100 g/mm2= 10 bars). The equivalent intensity indicates how the activation produced by

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PLAP peptide would equate to mechanical stimulation. Doses: 20 nmols, MH case 1; 37 nmols, MN case 4, 6, 7, 8 and 10 MH case 4; 75 nmols, MN cases 1, 2, 3, 5 and 9, MH case 2 and 3. P Prostaglandin E2, 5 serotonin, B bradykinin, H histamine, S saline, 21 21 amino acid PLAP peptide fragment, N negligible response, N T n o test made


Mean Transduction Equivalent activity range intensity (gz) (N/mm 2) (N/mm 2)

MN 1 18.90 S, 5, B, 21 17.50 13.20 0.90-1.63 0.92 2 10.40 5, P, B, H, 21 14.20 13.00 0.60-1.32 0.58 3 24.80 S, H, 21 5.34 5.34 NT - 4 18.20 S, B, 21 14.80 6.70 0.34-1.1() 0.79 5 23.00 B, 21 10.10 10.00 NT - 6 19.00 S, B, 21 0.08 0.08 0.29-1.56 N 7 27.70 S, B, M, 21 14.70 12.70 0.28-1.57 0.23 8 22.20 P, 5, B, 21 5.26 2.69 0.78-3.25 0.95 9 47.50 B, 5, 21 0.07 0.07 0.38-1.29 " N

10 - S, B, 21 22.40 14.90 NT - 11 23.00 5, P, B, H, 21 - - 0.50-1.07 - 12 39.60 5, B, 21 - - 0.21-1,02 - 13 28.30 P, 5, B, 21 -- -- 0.97-1.96 -- 14 11.10 S, H, 21 - - ' NT - 15 26.40 P, 5, B, 21 - - NT - 16 26.60 S, B, 5, P, 21 - - 0.20-1.57 -

MH 1 4.90 S, 21 8.60 8.10 0.28-0.53 0.33 2 10.70 H, 5, 21 6.20 6.20 0.07-0.93 N 3 14.00 S, 5, B, P, 21 11.30 9.20 NT NT 4 13.80 S, B, 21 13.50 13.10 0.09-0.49 0.35

to be nociceptors (intense pressure receptors) were not act ivated by melittin or P L A P peptide. It may be that despite low conduct ion velocity, high threshold, b road response range, and potent ial for sensitization (Cooper et al. 1991a), these afferents are not nociceptors. In animal preparat ions , physiological studies are impaired due to a lack of absolute classification criteria for nociceptive afferents. Regardless of reactivity to intense heat, intense mechanical, or chemical stimuli, definitive classification of an afferent as a nociceptor cannot be made. As a consequence, mixed popula t ions of afferents are routinely the subject of physiological investigations. This could account for the restricted popula t ion of nociceptors that are sensitive to classic algesics agents such as serotonin and bradykinin. On the other hand, there is no reason to expect that all nociceptor classes should be act ivated by melittin, P L A P peptide, or the classic algesic substances. Moreover , sensitivity of various classes to chemical agents may be greatly modified by inflamma to ry conditions.

Melittin is a powerful c o m p o u n d that induces production and release of m a n y pro- in f lammatory substances (Hassid and Levine 1977; Mollay and Kreil 1974; Salari et al. 1985; Shier 1979). At least some of these effects are mimicked by P L A P peptide (Bomalaski and Clark 1993; Clark et al. 1991). Therefore, act ivat ion of nociceptors by P L A P peptide and melittin could be media ted by the produc t ion of other agents. Al though it

cannot be said conclusively, this seems for the mos t par t unlikely.

Both melittin and P L A P are capable of producing a rapid accumulat ion of the products of phosphol ipase A2. However, the act ivat ion of M N s was not likely to arise f rom the format ion of PGEs. Extensive tests of the capacities of PGE1 and PGE2 to activate myelinated nociceptors proved futile in this report , as it has in m a n y others. In addition, mutua l affinity of PGI2 and PGE1 for the same receptors (Blair and M a c D e r m o t 1981; Coleman et al. 1984) further suggests that format ion of this eicosanoid was not likely to mediate the activity we observed. Nor does our data suggest that serotonin would part icipate in this act ivat ion and, moreover , it is only via a very indirect mechanism that one would expect the generation of serotonin.

As it is likely that LTB 4 and other products of the l ipoxygenase pa thway were produced by these peptides (Bomalaski et al. 1990; Salari et al. 1985), l ipoxygenase products could contr ibute to, or account for the activa- t ion that was observed. Al though we have yet to examine the capacity of LTB 4 to activate nociceptors, they and other dihydroxyeicosatetranoic acids have failed to be effective in this manne r in other prepara t ions (Madison et al. 1992; Mart in et al. 1987).

It is also possible that s imultaneous combinat ions of algesics (see Kessler et al. 1989; Meyer et al. 1991) or other untested or yet unrecognized agents can account

26

for the activation of these nociceptors. This cannot be determined without further study. Arguing, in general, against an indirect mediation is the fact that the activation was relatively rapid (immediate to 40 s delay), and permitted little time for the synthesis and release of mul- tiple compounds. Therefore, it is likely that the activation of mechanonociceptors by melittin or PLAP peptide is direct, specific, and possibly receptor mediated.

The manner in which PLAP peptide would interact with nociceptors is uncertain. Structural similarities between PLAP and melittin suggest that they may have similar modes of action. A variety of interactions between melittin and lipid membranes have been described. Some of these interactions suggest receptor activation. Like other insect venom components, melittin can activate G proteins (Higashijima et al. 1990). More- over, the presence of tachyphylaxis and apparent target- ing of specific afferent subclasses supports the presence of a melittin receptor. Alternately, nociceptor features - such as thin diameter and unmyelinated endings - may play a role in determining melittin or PLAP susceptibility.

Melittin is a highly amphipathic compound that as- sumes an alpha helical form in lipid membranes (Baten- burg et al. 1987; Kempf et al. 1982; Yiannakis et al. 1986). It is believed that structural features of the peptide produce a wedging action that disrupts membrane structure, and creates pores which permit the exchange of ions and large molecules with the extracellular space (DeGrado et al. 1982; Weissmann et al. 1969). Once formed, the pores may close, due to migration of the peptide into the cell. Potentially, the formation and dis- solution of melittin pores could create the bursting discharges which were observed as more substance diffused towards the ending and passed into the intra-axonal compartment. Myelination and receptor specializations would form barriers to membrane perturbations. There- fore, melittin (or PLAP) may be selectively effective on unmyelinated endings in the absence of a specific receptor.

In general, slowly conducting fibers were more consistently activated by melittin and PLAP peptide than rapidly conducting fibers (see Tables 2 and 3). While this might be due to the distribution of a putative melittin/ PLAP receptor, it might otherwise occur indirectly via a greater susceptibility of small diameter fibers to transient ionic fluxes. The likelihood of afferent activation following creation of transient pores could be expected to be increased in small diameter afferents where a greater ratio of membrane surface to intra-axonal volume would result in a proportionately greater impact of ionic fluxes on membrane potential. In this manner, melittin or PLAP may interact in the same manner with all afferent membranes (in the absence of a specific receptor), but would only be able to induce activation in small diameter fibers.

The capacity of melittin to disrupt membranes suggests that lysis could contribute to the incidence of activation. At high doses, in vitro, interaction of melittin

with cell membranes can result in lysis (Bomalaski et al. 1989; Weissmann et al. 1969; Yiannakis et al. 1986). It does not seem likely that lysis was the mechanism of melittin activation. As our afferents typically retained mechanical reactivity after melittin (24 of 27 MN, MH and C nociceptors), and continued discharging over long periods of time, it can be argued that a generalized toxic effect on fibers did not play a singular role; still, highly localized toxicity cannot be ruled out.

In contrast to melittin, the physiology and pharma- cology of PLAP is less well understood; nevertheless, there is a substantial indication that PLAP plays a fun- damental role in the cascade of events leading to the generation of eicosanoid mediators and the release of tissue damaging substances. PLAP is widely distributed amongst inflammatory and other cell types (T ceils, monocytes, macrophages, endothelial cells, synovio- cytes, chondrocytes, giant cells, and smooth muscle cells; Bomalaski and Clark 1993; Bomalaski et al. 1990). Both PLAP and PLAP peptide are potent activators of PLA2 in both T cells and smooth muscle cells (Boma- laski et al. 1992; Clark et al. 1987; Clark et al. 1988; Clark et al. 1991), and stimulate the release of superoxide radicals and lysosomal enzymes in neutrophils and monocytes (Bomalaski et al. 1989; Bomalaski et al. 1990). Moreover, PLAP synthesis is required to mediate the activation of PLA 2 by interleukin-1, tumor necrosis factor-alpha and leukotriene D4 in endothelial, smooth muscle, and T cells (Clark et al. 1988; Clark et al. 1991). It is not yet known whether PLAP plays a similar role in PLA2 activation in the other cells in which it has been identified; however, PLAP independent lines of PLA2 activation (via bradykinin) have also been demonstrated (Clark et al. 1991).

At this point, evaluation of the effectiveness of PLAP as an algesic is complicated by several factors. In the first case, it is yet to be determined whether PLAP is a single mediator or a precursor substance. Identification of the form of the active subunit(s) contained within a putative PLAP precursor molecule has obvious implica- tions for its effectiveness in any system. Limited avail- ability has restricted testing of PLAP in its 28 kDa form. The effectiveness of the PLAP peptide fragment as both an activator of PLA2 and nociceptors suggests that this fragment may be a naturally occurring endogenous mediator; the presence of dibasic peptides within the PLAP molecule indicate that this protein may be a prohormone that requires further processing to achieve full biological activity. This is further indicated by the re- cent suggestion that a portion of the PLAP sequence contains four copies of a G protein receptor subunit (Peitsch et al. 1993). This subunit is separated from a sequence containing the 21 amino acid PLAP fragment by dibasic and monobasic peptides that are potential points of attack by prohormone convertases (Hutton 1990; Sch/ifer et al. 1993), and it has been suggested that the PLAP peptide may be a regulatory molecule for this G protein subunit. If this proves to be the case, the active form of the peptide could be three times longer than

the 21 amino acid peptide used in this study. Moreover, it may not be a coincidence that one of the predicted sites of cleavage by prohormone convertases would produce a PLAP peptide that is, like melittin, 26 amino acids long.

The PLAP 21 peptide fragment is a powerful activator of PLA2, and is more effective, in vitro, than melittin in this regard (Clark et al. 1991). In contrast, this PLAP peptide fragment was much less effective than the 26 amino acid peptide, melittin, as an in vivo activator of A delta nociceptors. Several factors could influence the in vivo efficacy of PLAP peptide. The 21 amino acid PLAP peptide fragment was chosen as that peptide with a high sequence homology to melittin (Clark et al. 1991). As noted above, this might not be the optimal length for the peptide to express its full biological activity. In addition, secondary factors such as tissue pH, co-mediators, rate of catabolism and lipophilicity of PLAP peptides could play a role in determining its efficacy as an algesic. Much remains to be determined about this interesting pro-inflammatory protein.

Acknowledgements We would like to thank Monica Gonzalez and Thomas JUrek for their help with the data analysis. Special thanks goes to Martha Oberdorfer for preparatory surgery and the management of staff. We would also like to acknowledge the technical assistance of James Murphy, Peter Michel, T. Beaky, and Anwaral Azam for design and construction of equipment and software. Peptide synthesis was provided by the Protein Chem- istry Core Facility, Interdisciplinary Center of Biotechnology Re- search, University of Florida (NIH $10RR02496). This project was supported by NIH/NIDR DE 08701 and NIH AR 39382.

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