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A combined study of behavioural and electrophysiological tests was carried out in order to assess the role of metabotropic glutamate receptors (mGluRs) in mediating sensory inputs to the spinal cord of the rat. In the behavioural study the responses of conscious animals, with or without carrageenan-induced inflammation, to noxious mechanical and thermal stimuli were observed both before and after the intrathecal administration of mGluR antagonists L(+)-2-amino-3-phosphonopropionic acid (L-AP3) and (S)-4-carboxy-3-hydroxyphenylglycine (CHPG). It was found that the mGluR antagonist (S)-CHPG was capable of increasing both mechanical threshold and thermal latency in both groups of animals, and L-AP3 did so in those with inflammation induced in their hindpaw. Following this study, the responses of single lamina III-V dorsal horn neurons to an innocuous A beta fibre brush stimulus and a noxious C fibre (mustard oil) stimulus were extracellularly recorded and the effect of ionophoretically applied drugs was examined. Cyclothiazide (CTZ), a selective antagonist at mGluR1, markedly reduced the activity evoked by mustard oil, but not that elicited by brushing of the receptive field. Activity induced in dorsal horn neurons by ionophoresing various mGluR subgroup agonists was examined. CTZ successfully inhibited the activity evoked by group I mGluR agonist 3,5-dihydroxyphenylglycine (DHPG). In comparison to the neurons which responded to the ionophoresis of DHPG, less were activated by the selective mGluR5 agonist trans-azetidine dicarboxylic acid (t-ADA). Together these results indicate that group I mGlu receptors, in particular mGluR1, play a crucial role in mediating nociception, particularly following a sustained noxious input.

We have previously described the changes in spinal cord neuropeptides in the unilateral sciatic chronic constriction injury (CCI) model of Bennett and Xie [Pain, 33 (1988) 87-108] at 28 days, a time of maximum mechanical hyperalgesia. In this study we examine the same model 100-120 days post injury by which time resolution of the hyperalgesia and peripheral nerve injury has occurred according to previous studies. Rats underwent either CCI of the sciatic nerve (n = 12) or else sham operation (n = 8) which involved exposure but no ligation of the nerve. Mechanical hyperalgesia was assessed with a Ugo-Basile analgesymeter and immunohistochemistry performed on the spinal cord sections of the animals and quantified using a confocal microscope. At this late time point CCI rats were no longer significantly mechanically hyperalgesic compared to the sham animals (P > or = 0.09). However, examination of the lumbar spinal cord revealed the following changes. (i) The neuropeptides substance P (SP) (P < 0.0001) and galanin (P < 0.003) both showed decreases of about 30% ipsilaterally in immunoreactivity in laminae 1 and 2 of the dorsal horn compared to the sham operated animals. (ii) Calcitonin gene-related peptide (CGRP) and neuropeptide Y (NPY) in laminae 1 and 2 showed no significant changes compared to sham animals. (iii) NPY levels in laminae 3 and 4 of the spinal cord showed a 15% increase in immunoreactivity compared to sham animals (P = 0.008). These results indicate that changes in neuronal markers in the spinal cord can persist after apparent resolution of a peripheral nerve injury. We suggest that these changes may form a substrate for subsequent development of abnormal pain states.

The introduction of 5-HT3 antagonists, such as ondansetron, as antiemetic agents has transformed the management of patients receiving chemotherapy or radiation therapy. Studies in animal models with NK1 antagonists suggest that these represent a new class of antiemetic agents having a broader spectrum of activity than 5-HT3 antagonists. Compounds of this class may prove to be more effective in man against delayed emesis induced by cisplatin, post-operative nausea and vomiting and motion sickness. Thus, they have the potential to complement 5-HT3 antagonists and so provide a further advance in the management of nausea and vomiting.

In the ferret, 5-HT3 receptor antagonists are effective in controlling emesis produced by cytotoxic agents or radiation. To investigate the possibility that substance P has a role, as well as 5-HT, in the emetic reflex pathway, we have examined the anti-emetic effects of a NK1 receptor antagonist (racemic CP-99,994) in the ferret. Racemic CP-99,994 was effective against a range of emetogens, comprising cytotoxic drugs, radiation, morphine, ipecacuanha and copper sulphate.

1. Intracellular pH was recorded (double-barrelled pH-selective microelectrodes) in single ventricular myocytes and whole papillary muscles isolated from guinea-pig heart. Both preparations were acid-loaded by various manoeuvres (addition and removal of external NH4Cl or CO2) in order that a comparison could be made of the size and speed of intracellular pH changes and hence of the apparent intracellular buffering power (beta). 2. For the same acid-loading procedure, the size of intracellular pH (pHi) changes was about threefold larger in the isolated myocyte than in whole papillary muscle. The rate of initial acid loading as well as the subsequent rate of pHi recovery (caused by acid extrusion from the cell) were also threefold faster in the myocyte. Estimates of apparent intrinsic (non-CO2) buffering power, based upon the size of pHi changes during acid loading, were 15-20 mmol l-1 for the myocyte and about 70 mmol l-1 for whole muscle. This latter value is similar to previous estimates of beta in heart. 3. When acid extrusion was reduced by applying a high dose of amiloride (1 mmol l-1), then the size of the pHi change during acid loading increased greatly in papillary muscle but changed much less in the myocyte; beta now appeared to be about 30 mmol l-1 in whole muscle but remained essentially unchanged in the myocyte. 4. We conclude that previous values for beta in cardiac muscle have been greatly overestimated because of the presence of sarcolemmal acid extrusion. Paradoxically, this error in estimating beta is far less evident in the isolated myocyte. We suggest that this is because a much more rapid acid loading is achievable in the myocyte so that acid loading will be blunted less by acid extrusion than in whole muscle. We present a simple mathematical model that demonstrates this phenomenon. We conclude that beta in ventricular muscle is likely to resemble that measured in the isolated myocyte, i.e. 15-20 mmol l-1. 5. Slow acid loading in whole ventricular muscle will also affect the kinetics of pHi changes. The model indicates that the rate of pHi recovery from an acid load in papillary muscle does not reflect the pHi dependence of acid extrusion. Instead, it is heavily influenced by the slow rate of acid loading. This emphasises that great care should be taken when interpreting the kinetics of pHi changes in multicellular ventricular preparations.

Measurements of membrane potential and resistance have been made in zona-free eggs of mice and hamsters. The mean +/- S.D. values for membrane potential were -91 +/- 28 mV (mouse) and -97 +/- 29 mV (hamster) and for input resistance were 430 +/- 230 M omega (mouse) and 410 +/- 150 M omega (hamster) respectively. Large fluctuations (20 mV) of membrane potential occurred apparently at random and these were accompanied by changes of membrane resistance. Depolarizing current pulses passed through the recording micro-electrode evoked action potentials in eggs of both species. The threshold for excitation was about -50 mV, the maximum rate of rise of the action potential was about 3 V.s-1 and its peak value was about +13 mV. Action potentials could be evoked in eggs bathed in sodium-free solution or in normal solution containing tetrodotoxin (3 microM). The presence of cobalt (5-20 mM), lanthanum (1 mM) or verapamil (200-400 microM) in the bathing solution suppressed the action potential. Raising the extracellular calcium concentration from 4 to 40 mM increased the peak value of the action potential by 25 mV. It is concluded that the plasma membranes of mouse and hamster eggs have voltage-dependent calcium channels.

Microelectrode recordings from cells in rabbit ciliary epithelium have been made in vitro. Ionophoresis of Lucifer Yellow dye from microelectrodes during measurements of potential confirmed that the recordings were intracellular. Dye passed from the impaled cells into adjacent cells in both the nonpigmented and pigmented layers of the epithelium. Electrical coupling between epithelial cells also was observed. The mean (+/- SD) values of the potential measured across the basolateral membranes of the nonpigmented cells was -65 +/- -15 mV (n = 77); the mean value of the input resistance at this intracellular recording site was 37 +/- 28 M omega (n = 17). The membrane potential was reduced by raising the concentration of extracellular potassium but unaffected by changes in the concentrations of sodium, chloride, or bicarbonate ions. After a period of deprivation of extracellular potassium, the cells hyperpolarized without a measurable change in membrane resistance when potassium was restored to the bathing solution; this transient response to potassium was abolished by preincubation with ouabain or by bathing the epithelium in a solution lacking sodium. It was concluded that the ciliary epithelial cells are permeable to potassium but exhibit only a low permeability to sodium, chloride, or bicarbonate ions; that the cells possess an electrogenic Na/K pump; and finally, that all of the cells in the epithelium function as a syncytium.

Cell-attached patch recordings were made from zona-free mouse eggs to examine currents at the single-channel level; two types were identified. A channel with a mean conductance of 130 pS showed changes in reversal potential on dilution of the patch pipette solution consistent with a cation channel; the channel current persisted when the only cation in the pipette was K+: it was concluded that this channel was permeable to K+. The binomial distribution was used to describe the proportion of time 0, 1, 2, ..., n channels were open in multichannel recordings of the K+ channel. Bursts of inward currents of about 1 pA, which showed voltage activation and could be recorded in the presence of CsCl and tetraethylammonium (TEA), were identified as Ca2+ channels.

1. Intracellular pH (pHi) and Na+ activity were recorded (ion-selective microelectrodes) in guinea-pig papillary muscle and the sheep cardiac Purkinje fibre while simultaneously recording twitch tension. The effects of intracellular acidosis and alkalosis upon contraction were investigated. 2. A fall of pHi produced by reducing pHo was associated with a fall of twitch tension. Similarly, a rise of pHi produced by raising pHo produced a rise of twitch tension. The time course of the changes in tension correlated with the time course of changes of pHi rather than pHo. These results are consistent with previous work showing that acidosis inhibits contraction and that the inhibition depends upon a fall of pHi. 3. Changes of pHi were produced while maintaining pHo constant at 7.4. Removal of NH4Cl or addition of sodium acetate (pHo 7.4) reduced pHi but this gave either an increase of tension (papillary muscle) or an initial fall followed by a subsequent recovery of tension (Purkinje fibre). The increase or recovery of tension occurred despite the fact that there was an intracellular acid load. Thus, reducing pHi at constant pHo can increase tension whereas reducing pHi at low pHo (6.4, see paragraph 2) inhibits tension. 4. The increase of recovery of tension during intracellular acidosis produced at a constant pHo (7.4) was associated with a rise of intracellular sodium activity (aiNa). Amiloride (1.5 mmol/l), an inhibitor of Na(+)-H+ exchange, prevented the rise of aiNa during intracellular acidosis and also prevented the recovery of tension. It is concluded that the increase or recovery of tension at low pHi is secondary to a rise of aiNa caused by stimulation of Na(+)-H+ exchange. A rise of aiNa will elevate Ca2+ via sarcolemmal Na(+)-Ca2+ exchange and thus will elevate tension. 5. An intracellular acidosis produced by reducing pHo (6.4) does not elevate aiNa in the Purkinje fibre. In papillary muscle, aiNa rises but this occurs slowly and the rise is 50% smaller than that seen when the same intracellular acidosis is induced at normal pHo (7.4). The net depression of tension under these conditions thus correlates with the lack of a large rise of aiNa. 6. Knowing the quantitative dependence of tension upon both aiNa and pHi in the two tissues it is possible to predict the recovery of twitch tension during intracellular acidosis at constant pHo (7.4), using the changes of pHi and aiNa measured under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Measurements have been made of membrane potential and input resistance of zona-free hamster eggs from single micro-electrode recordings. At room temperature (20-23 degrees C) the mean (+/- S.D.) values for the potential and resistance were -30 +/- 8 mV and 280 +/- 130 M omega (n = 94 eggs). At 37 degrees C the mean (+/- S.D.) values for the potential and resistance were -39 +/- 13 mV and 230 +/- 60 M omega (n = 60 eggs). The most negative potential recorded at room temperature was -51 mV in a cell which had an input resistance of 620 M omega. At 37 degrees C six eggs out of sixty had potentials more negative than -50 mV and three of these gave all-or-none action potentials in response to depolarizing current pulses. In a separate series of experiments with high resistance micro-electrodes (ca. 100 M omega) six eggs out of twenty-one had potentials more negative than -50 mV and four of these were electrically excitable. Transient potential recordings during impalement indicated that the potential was more negative than -30 mV but that the insertion of a micro-electrode produced a leak pathway with a resistance of about 10 M omega, substantially smaller than the steady-state estimates of the input resistance (see above). Whole-cell recordings with patch pipettes gave potentials in the range -30 to -80 mV and input resistances in the range 180 to 350 M omega (n = 8); four eggs gave action potentials in response to depolarizing current pulses passed through the patch pipette. It is concluded that the leak impalement artifact is so significant in micro-electrode recordings from hamster eggs that it prevents routine reliable potential measurements.

Intracellular pH (pHi) and Na (aina) were recorded in isolated sheep cardiac Purkinje fibres using ion-selective microelectrodes while simultaneously recording twitch tension. A fall of pHi stimulated acid-extrusion via sarcolemmal Na-H exchange but the extrusion was inhibited by reducing extracellular pH (pHo), indicating an inhibitory effect of external H ions upon the exchanger. Intracellular acidosis can reduce contraction by directly reducing myofibrillar Ca2+ sensitivity. The activation of Na-H exchange at low pHi can offset this direct inhibitory effect of H+ ions since exchange-activation elevates aina which then indirectly elevates Ca2+i (via Na-Ca exchange) thus tending to restore tension. This protection of contraction during intracellular acidosis can be removed if extracellular pH is also allowed to fall since, under these conditions, Na-H exchange is inhibited.

Measurements of membrane potential and resistance have been made in zona-free hamster eggs. The resting potential lay in the range -9 to -100 mV and the input resistance fell in the range 14 to 440 M omega; high resting potentials were associated with large input resistances. Calcium injected ionophoretically into an egg from an intracellular micro-electrode caused a reduction of the membrane resistance. The estimated reversal potential for the calcium-evoked response was about -80 mV and its amplitude depended on the extracellular concentration of potassium but not on the chloride concentration. We conclude that membrane potassium channels open in response to a rise in the cytosolic concentration of calcium ions. Evidence is presented to suggest that micro-electrode recordings of the membrane potential and resistance of eggs suffer from an impalement leak artifact. The presence of the artifact lowers the resting potential and resistance of the cell so that intracellular calcium injection causes a hyperpolarization. We conclude that a hyperpolarizing response to calcium would be unlikely in the absence of an impalement artifact.

1. The mechanism of the rate-dependent decrease in intracellular pH (pHi) and its recovery were studied in isolated sheep cardiac Purkinje fibres. Intracellular Na+ activity (aiNa) and pHi were measured using ion-selective microelectrodes. Twitches were elicited by field stimulation or by depolarizing pulses applied using a two-microelectrode voltage clamp. 2. A 3 Hz train of short (50 ms) depolarizing voltage-clamp pulses induced a reversible fall in pHi which was accompanied by a reversible increase in aiNa. A train of longer (200 ms) pulses also produced a fall in pHi which was now paralleled by a decrease in aiNa. These observations indicate that the rate-dependent acidosis is not dependent upon a rise in aiNa. 3. Neither the fall in pHi nor the increase in aiNa seen upon an increase in action potential frequency was inhibited by amiloride (1 mmol l-1) which indicates that Na+-H+ exchange is not involved in the generation of the acidosis. Furthermore, the rate-dependent acidosis was not abolished in Na+-free solution (Li+ or N-methyl glucamine substituted) indicating that other Na+-requiring processes (such as Na+-Ca2+ exchange) are not a necessary requirement. Rate-dependent pHi changes were also unaffected by the stilbene compound DIDS indicating no participation by Cl--HCO-3 exchange. 4. The rate-dependent acidosis was inhibited by the organic calcium antagonist D600 (20 mumol l-1) which also inhibited twitch tension. This suggests that the acidosis is related to the activation by Ca2+ of developed tension. D600 also inhibited the rate-dependent rise in aiNa (field stimulation). 5. The rate-dependent acidosis was not inhibited by cyanide (2 mmol l-1) but it was blocked by iodoacetate (0.5 mmol l-1) and by 2-deoxyglucose (DOG) (10 mmol l-1, applied in glucose-free solution). These results suggest that the acidosis is generated metabolically via stimulation of glycolysis, following an increase in contraction. Contributions from aerobic metabolism are likely to be small. 6. Twitch tension was inhibited by ryanodine (10 mumol l-1) but the drug had little inhibitory effect on the rate-dependent acidosis. A tonic component of tension was observed, however, in the presence of ryanodine. The lack of effect of ryanodine upon the rate-induced acidosis is discussed. 7. The half-time of pHi recovery from the frequency-dependent acidosis was consistently shorter than that from an intracellular acid load induced by adding and then removing external NH4Cl (10 mmol l-1).(ABSTRACT TRUNCATED AT 400 WORDS)

At room temperature micro-injections of calcium or strontium produced transient hyperpolarizations with an associated rise in input conductance. By contrast micro-injections of potassium, barium, magnesium, cobalt or lanthanum did not produce hyperpolarizations. The reversal potential for the hyperpolarizing response was about -80 mV. Some responses to calcium injections appeared to suffer from an additional transient leak conductance generated by the injected current. In these cases the recovery of the potential and the conductance to normal values was prolonged. The reversal potential of this additional leak pathway was about -10 mV. Experiments designed to investigate the role of active calcium extrusion from the cells showed that extracellular lanthanum or quercetin caused a pronounced slowing of the recovery phase of the potential and conductance response to calcium injection. The metabolic uncoupler dinitrophenol also prolonged the calcium-evoked responses. The replacement of extracellular sodium by lithium or choline produced no alteration in the time course of the calcium-evoked responses, thus suggesting that sodium-calcium exchange exerts no rate control on the recovery phase of those responses.

1. The influence of repetitive activity upon intracellular pH (pHi), intracellular Na+ activity (aNA(i)) and contraction was examined in isolated sheep cardiac Purkinje fibres. Ion-selective microelectrodes were used to measure intracellular Na+ and H+ ion activity. Twitch tension was elicited by field stimulation or by depolarizing pulses applied using a two-microelectrode voltage clamp. Experiments were performed in HEPES-buffered solution equilibrated either with air or 100% O2. 2. An increase in action potential frequency from a basal rate of 0.1 to 1-4 Hz induced a reversible fall in pHi and a reversible rise in aNa(i). These effects reached a steady state 3-10 min following an increase in stimulation frequency, and showed a linear dependence on frequency with a mean slope of 0.023 pH units Hz-1 and 0.57 mmol l-1 Hz-1, respectively. The rise in total intracellular acid and aNa(i) associated with a single action potential was estimated as 5.3 mu equiv l-1 of acid and 3.5 mu equiv l-1 of Na+. 3. At action potential frequencies greater than 1 Hz, the rate-dependent rise in aNa(i) was usually accompanied by a positive force staircase. 4. The fall in pHi following a rate increase also occurred when fibres were bathed in Tyrode solution equilibrated with 23 mM-HCO3- plus nominally 5% CO2/95% O2. In these cases, however, the fall in pHi was halved in magnitude. 5. In fibres exposed to strophanthidin (0.5 microM), the rate-dependent fall in pHi was doubled in magnitude and its time course was more variable than under drug-free conditions. The rate-dependent rise in aiNa was also usually larger in strophanthidin. 6. In order to examine the influence of the rate-dependent acidosis on developed tension, the acidosis was reversed experimentally by adding 2 mmol l-1 NH4Cl to the bathing solution. This produced a rise in pHi accompanied by a large increase in twitch tension. Such an effect of pHi upon tension was quantitatively similar to that observed in previous work on Purkinje fibres (Vaughan-Jones, Eisner & Lederer, 1987). 7. It is concluded that the rate dependence of pHi will influence both the magnitude and the time course of an inotropic response to a change in heart rate.