Isolated Rabbit Ear Artery Research Articles

Quantitative analysis of the agonist and antagonist actions of some ATP analogues at P2X‐purinoceptors in the rabbit ear artery

1. The agonist and antagonist effects of a series of beta, gamma-methylene dihalo- and 2-methylthio-substituted analogues of ATP at P2x-purinoceptors have been analysed on the rabbit isolated ear artery preparation. Cumulative and sequential dosing experimental protocols were employed in the construction of agonist concentration-effect curves in order to address the possible influence of acute receptor desensitization on subsequent analyses. 2. Using the cumulative curve design the following results were obtained: D-AMP-PCBr2P, 2-methylthio-D-AMP-PCCl2P, L-AMP-PCF2P, L-AMP-PCCl2P and LAMP-PCBr2P each behaved as partial agonists. D-AMP-CPP was used as a reference full agonist and these analogues were analysed by the comparative method of Barlow et al. (1967), to provide estimates of affinity and efficacy. 2-Methylthio-L-AMP-PCBr2P was virtually silent as an agonist and was analysed as a competitive antagonist by Schild analysis. 3. Two agonists, L-AMP-PCCl2P and L-AMP-PCBr2P, were analysed by the sequential curve design, and the antagonist effects of one of the agonists, L-AMP-PCBr2P were also analysed using this protocol. The resulting estimates of affinity and efficacy, while similar to those obtained with the cumulative design, indicated that acute desensitization may affect curve definition and estimation of these quantities. 4. The following structure-activity trends emerged: D-analogues tended to have higher efficacy but lower affinity than L-analogues; efficacy varied markedly and inversely with the atomic weight of the halogen while affinity was only minimally affected; 2-methylthio- substitution also reduced efficacy with minimal effect on affinity. 5. The results of this analysis are discussed in terms of the utility of affinity and efficacy information in the classification of purinoceptors and the design of chemical probes for them.

Phenylephrine-induced rhythmic activity in the rabbit ear artery.

Isolated rabbit ear arteries displayed rhythmic contractions when stimulated with alpha 1 agonist phenylephrine. These rhythmic responses were greatly attenuated by endothelium removal. However, contractions were sufficiently rhythmic in the arteries treated with NG-monomethyl-L-arginine, NG-nitro-L-arginine and indomethacin, synthetic inhibitors of endothelium-derived nitric oxide and prostanoids. Phenylephrine-induced rhythmic contractions were converted to tonic contractions by the blockade both of the voltage-dependent Ca2+ channel and the Ca(2+)-activated K+ channel by nifedipine and charybdotoxin, respectively. In contrast, glibenclamide, an ATP-sensitive K+ channel antagonist, did not alter the rhythmic contractions. These results suggest that endothelium may in part regulate the phenylephrine-induced rhythmic contractions in the rabbit ear artery, although endothelium-derived nitric oxide or prostanoids may not be involved in these responses. These endothelium-involved rhythmic responses may be attributed to the activation of the voltage-dependent Ca2+ channel and the Ca(2+)-activated K+ channel.

Intraarterial propofol is not directly toxic to vascular endothelium.

To determine if accidental intraarterial injection of propofol results in vascular damage, the effect of bolus administration of propofol on vascular smooth muscle and the endothelium was investigated using the isolated rabbit ear artery. Ear artery segments, removed from urethane anesthetized rabbits, were perfused with Krebs solution (1 ml.min-1) and pressurized to 60 mmHg before being constricted with extraluminal norepinephrine (1.8-4.2 x 10(-6) M). The external diameter of the vessel was measured by an array of light-dependent diodes. Functional responsiveness was determined by the degree of constriction to norepinephrine and the subsequent dilatation of the artery to intraluminal acetylcholine (2 x 10(-6) M) and glyceryl trinitrate (2 x 10(-6) M), and by the myogenic reactivity to a pressure increase from 60 to 100 mmHg. These responses were measured before and after perfusion with 1% propofol for 120 s. Administration of propofol did not result in any vasoactivity nor did it increase the sensitivity to norepinephrine. Vessels maintained their capacity to dilate to both agents, while the myogenic activity was unaffected. Histologic examination of the propofol exposed vessels showed no changes to smooth muscle structure, and the endothelial layer remained intact.

Prejunctional muscarine receptors in the rabbit ear artery differ from M1, M2 and M3 muscarine receptors.

The ability of several selective muscarine receptor antagonists to inhibit the effect of carbachol on prejunctional muscarine receptors on sympathetic nerve endings in the rabbit isolated ear artery was investigated to characterise the receptor subtype involved. Carbachol did not reduce responses to exogenous noradrenaline and the inhibitory effect of carbachol on responses to nerve stimulation was unaffected by hexamethonium (10 microM) indicating that the effect of the muscarine agonist was exerted prejunctionally and was not modulated by nicotine receptor stimulation. The dissociation constants or apparent dissociation constants obtained using (+/-)-benzhexol (pKB; 6.63), (R)-benzhexol methiodide (8.11), dicyclomine (5.86), (+/-)-telenzepine (7.34), AF-DX 116 (6.95), himbacine (7.60), (+/-)-hexahydrosiladiphenidol (5.39) and a bisquaternary ammonium compound, heptane-1,7-bis(dimethyl-3'-phthalimidopropyl ammonium bromide) (5.84), indicate that the muscarine receptor subtype involved is not of the M1, M2 or M3 subtype.

O-421 - Characterization of relatively immediate relaxant effects of cholera toxin on the isolated rabbit ear artery

O-421 - Characterization of relatively immediate relaxant effects of cholera toxin on the isolated rabbit ear artery

Modulation of noradrenaline‐induced membrane currents by papaverine in rabbit vascular smooth muscle cells.

1. The modulation of noradrenaline-induced membrane currents by papaverine was studied with the nystatin whole-cell technique in freshly isolated rabbit ear artery and portal vein cells. 2. At a holding potential of -50 mV (in potassium-free conditions), papaverine inhibited the Ca(2+)-activated inward chloride current ICl(Ca) evoked by ionophoretic application of noradrenaline in rabbit portal vein cells in a concentration-dependent (10(-5)-10(-4) M) manner. 3. With potassium-containing solutions (at 0 mV) 10(-4) M-papaverine reversibly abolished the Ca(2+)-activated outward potassium current IK(Ca) induced by noradrenaline in both protal vein and ear artery cells. Occasionally, 10(-4) M-papaverine itself activated IK(Ca) before it abolished the response to noradrenaline. 4. Higher concentrations of papaverine (up to 10(-3) M) always evoked both ICl(Ca) and IK(Ca) at -50 and 0 mV respectively, before it abolished these responses to noradrenaline in both ear artery and portal vein cells. In the presence of 10(-3) M-papaverine higher doses of noradrenaline evoked the non-selective cation current in some portal vein cells. 5. In the presence of 10(-2) M-caffeine, which elicited both IK(Ca) and ICl(Ca), papaverine (10(-3) M) failed to activate either current and 10(-3) M-papaverine reduced the caffeine-induced IK(Ca) by 94%. In contrast, IK(Ca) induced by the calcium ionophore ionomycin was unaffected by papaverine. In Ca(2+)-free, 1 mM-EGTA external solutions, 10(-3) M-papaverine still induced IK(Ca) and blocked the response to noradrenaline. 6. The alpha 1-adrenoceptor antagonist prazosin (5 x 10(-8) M) completely blocked the noradrenaline-induced IK(Ca) but currents evoked by papaverine (10(-3) M) were unaffected by prazosin. 7. It was concluded that papaverine modulated noradrenaline-induced membrane currents in vascular smooth muscle cells by releasing and depleting caffeine-sensitive intracellular Ca2+ stores.

Endothelium-dependent relaxation induced by sodium fluoride in the rabbit ear artery

Sodium fluoride (NaF) produced concentration-dependent relaxation of isolated rabbit ear artery precontracted with norepinephrine. In contrast, an arterial preparation with the endothelium rubbed off did not relax, but contracted in response to NaF. NaF-induced relaxation was not influenced by indomethacin but was inhibited by methylene blue or NG-monomethyl-L-arginine. The results indicate that NaF relaxes the artery by releasing a so-called EDRF.

Effects of Suramin on the ATP- and α,β-Methylene-ATP-induced Constriction of the Rabbit Ear Artery

Abstract The isolated rabbit ear artery contains both dilation-mediating P2Y-receptors and constriction-mediating P2X-receptors. Suramin antagonizes the effects of ATP at either receptor.

Lack of presynaptic modulation by isoprenaline of 3H-noradrenaline release from rabbit isolated ear artery

The aim of the present investigation was to examine whether or not presynaptic facilitatory beta-adrenoceptors are detectable on the postganglionic nerves in the rabbit isolated ear artery. Strips of rabbit central ear artery were incubated with 3H-noradrenaline (10(-7) mol/l; 30 min or 10(-6) mol/l; 60 min). Subsequently, they were washed repeatedly with physiological salt solution. The strips were subjected to electrical-field stimulation (S1-S8) and the resultant 3H-overflow was determined. When the ear artery was stimulated with 150 pulses (0.5 ms; 3 Hz; 225 mA), isoprenaline (10(-9)-10(-6) mol/l) either alone or in the presence of either rauwolscine (10(-6) mol/l) or phentolamine (10(-6) mol/l) did not alter the stimulation-evoked 3H-overflow. This was also the case in the presence of rauwolscine (10(-6) mol/l) plus either the selective phosphodiesterase inhibitor ICI 63 197 (3 x 10(-5) mol/l) or forskolin (10(-6) mol/l). When the ear artery was stimulated with 300 pulses (1 ms; 5 Hz; 225 mA), isoprenaline had no effect on the stimulation-evoked 3H-overflow. This was also the case when phentolamine (10(-6) mol/l) was present. Propranolol (10(-7)-10(-5) mol/l) did not alter the stimulation-evoked 3H-overflow. In some experiments, the stimulation current was reduced to 175 mA in order to obtain similar reference release (S3) values despite the presence of rauwolscine (150 pulses; 0.5 ms; 3 Hz). Even then, isoprenaline (10(-9)-10(-6) mol/l) did not change stimulation-evoked 3H-overflow. The results suggest that postganglionic sympathetic nerves in rabbit central ear artery do not possess presynaptic facilitatory beta-adrenoceptors.

Differential effects of calcium antagonists and Bay K 8644 on contractile responses to exogenous noradrenaline and adrenergic nerve stimulation in the rabbit ear artery

1. The effects of three calcium antagonists (nifedipine, verapamil, diltiazem) and the calcium agonist Bay K 8644 were compared on contractile responses of similar amplitude elicited by noradrenaline (NA) and electrical nerve stimulation (ENS) in the rabbit isolated ear artery. 2. Contractions induced by both NA (3 x 10(-7) M) and ENS (10 Hz, 10s) were almost exclusively mediated by alpha 1-adrenoceptors, since 10(-7) M prazosin abolished (NA) or almost abolished (ENS) the responses, and prazosin was more than three orders of magnitude more potent than rauwolscine on both types of response. 3. ENS-induced contractions were considerably less inhibited by nifedipine, verapamil and diltiazem than were those elicited by NA. Bay K 8644 enhanced responses to NA more than those to ENS. 4. The inhibitory effect of nifedipine and Ca2+ deprivation on NA-induced contractions decreased with increasing NA concentration. Reduction of the NA response by prazosin or phenoxybenzamine increased the nifedipine inhibition. 5. Reduction of the ENS-induced contractions by prazosin or phenoxybenzamine, or by use of a lower stimulation frequency did not increase the inhibitory effect of nifedipine. 6. In conclusion, the differential effects of the calcium antagonists on NA- and ENS-induced contractions were not related to differences in alpha-adrenoceptor subtype (alpha 1/alpha 2), receptor reserve or response amplitude, but may rather reflect temporal and spatial differences in alpha-adrenoceptor activation between the responses.

Characterization of P2x‐receptors in rabbit isolated ear artery

1. The isolated central ear artery of the rabbit contracts in response to adenosine 5'-triphosphate (ATP) and analogues, effects proposed to be mediated by stimulation of P2x-receptors. We have extended the characterization of the purinoceptor in this tissue by examining the effects of a series of receptor agonists. The study was designed in such a way as to avoid factors which normally limit attempts to classify receptors on the basis of agonist potency orders. 2. D-alpha, beta-methylene ATP (D-alpha, beta-meATP), D-beta, gamma-methylene ATP (D-beta, gamma-meATP), L-beta, gamma-methylene ATP (L-beta, gamma-meATP), 2-methylthio-D-ATP (2-MeSATP) and ATP produced concentration-related contractions of the ear artery with similar maximum responses, suggesting that they were full agonists. Selective desensitization of P2x-receptors abolished or greatly reduced responses to D-alpha, beta-meATP, L-beta, gamma-meATP, D-beta, gamma-meATP. Responses to ATP were inhibited by by desensitization but a significant resistant component was still apparent. 3. D-alpha, beta-meATP was the most potent agonist tested (pA50 6.47 +/- 0.04) being 2138 times more potent than ATP and approximately 9 times more potent than L-beta, gamma-meATP. The agonist potency order was: D-alpha, beta-meATP greater than L-beta, gamma-meATP greater than D-beta, gamma-meATP greater than or equal to 2-MeSATP greater than ATP. This is generally consistent with the order proposed for P2x-receptors. The relative potencies of P2x-agonists in the rabbit ear artery show both similarities to and differences from data obtained in other smooth muscle preparations.

Suramin is a slowly‐equilibrating but competitive antagonist at P2x‐receptors in the rabbit isolated ear artery

1. The antagonist dynamics of suramin were investigated at P2x-receptors in isolated rings of endothelium-denuded ear artery from New Zealand White (NZW) rabbits. 2. alpha, beta-Methylene adenosine 5'-triphosphate (ATP) concentration-effect curves were constructed cumulatively in a paired curve design in the absence and presence of increasing concentrations of suramin, incubated for 45 min. The slope of the resulting Schild plot was significantly greater than unity (1.50 +/- 0.08). 3. Assuming that slow equilibration by suramin explains the steep Schild plot, further experiments were conducted using short (15 min) and long (3 h) incubation times. The resulting Schild plot slopes were 1.66 +/- 0.36 and 1.06 +/- 0.13 respectively confirming the assumption. However, after 3 h incubation, suramin also caused depression of alpha, beta-methylene ATP curves. 4. In an attempt to minimize the depressant effect of suramin, a kinetic study was designed to calculate the minimum incubation times for each concentration of suramin used in the Schild analysis to achieve effectively complete equilibrium. Theoretically fractional occupancy for the antagonist is given by (r - 1)/r, where r is the dose-ratio. A plot of (r - 1)/r against time allowed the apparent 'on' and 'off' rate constants to be calculated. 5. With the resulting rate constant estimates, an optimised antagonism study was carried out in which incubation times were chosen such that greater than 95% occupancy by suramin could be achieved without agonist curve depression at each concentration of suramin used. 6. Under these conditions, suramin fulfilled all criteria for simple competition: parallel rightward displacement of alpha,beta-methylene ATP curves and a Schild plot slope of unity (1.00 + 0.09). The resulting pKB estimate was 4.79 + 0.05. This estimate of affinity was shown to be independent of the agonist used in another experiment in which L-beta-methylene ATP was employed (pKB = 5.17). 7. Under the same conditions, suramin was found to have no effect on KCI-induced contractions and only slight effects on phenylephrine- and histamine-induced responses.8. This analysis provides the first evidence that suramin is a genuine competitive P21-receptor antagonist.

Mechanisms behind the Relaxing Effect of Furosemide on the Isolated Rabbit Ear Artery

The effect of furosemide on isometric contraction and 86Rb uptake were studied in the isolated rabbit central ear artery (CEA). A concentration-dependent relaxing effect of furosemide (0.06 mM-1.0 mM) was found in vessel segments with intact endothelium. The maximal relaxation was 28.6 +/- 3.9% (10). The effect was not diminished in segments deprived of endothelium, and removal of endothelium itself caused no change of the force development to electrical field stimulation. The relaxing effect was time-dependent and stimulation-dependent and was not significantly affected by membrane depolarization induced by increasing external [K+] from 10 to 120 mM. The 86Rb uptake was inhibited by both furosemide and ouabain (8.0 +/- 0.5(8) and 5.3 +/- 0.5(8) versus 12.8 +/- 0.9(16) nmol (K+).mm-1.(10 min.)-1 in the furosemide (1.0 mM), ouabain (1.0 mM) and control groups, respectively) without interaction between the two drugs. The 86Rb uptake was not further inhibited by increasing the furosemide concentration from 0.12 mM to 1.0 mM. Our results suggest: firstly, the direct relaxing effect of furosemide on isolated vessel segments is endothelium-independent and secondly, the inhibition of the Na(+)-K(+)-Cl- cotransport and a possible consequent hyperpolarization of the membrane is unlikely to be the sole mechanism responsible for the vasorelaxant effect of furosemide. The demonstrated direct effect on vascular tone may be of clinical importance in situations with very high plasma concentrations of the drug or very low concentrations of serum albumin.

Release of 3H‐Noradrenaline from Rabbit Isolated Ear Artery

The stimulation-evoked 3H-overflow from rabbit isolated ear arteries preincubated with 3H-noradrenaline was studied. Three strips were derived from each central artery. The strips were incubated (30 min.) with 3H-noradrenaline (10(-7) M) and the spontaneous 3H-outflow and stimulation-evoked 3H-overflow were followed by fractional collection. After a wash-out period (75 min.), the strips were stimulated (225 mA; 150 monophasic pulses; 3 Hz; 0.5 msec.) several times. The initial stimulation-evoked 3H-overflow (S1) was about 5-fold higher than the subsequent five 3H-overflows (S2-S6) which remained almost constant. Bretylium (10(-5) M), tetrodotoxin (10(-6) M), and omission of Ca2+ from the physiological salt solution reduced the stimulation-evoked 3H-overflow by maximally 52%, 77% and 62%, respectively. An increase in stimulation current from 50 to 225 mA caused a continuous rise in stimulation-evoked 3H-overflow, which tended to be Ca2(+)-sensitive. The stimulation-evoked 3H-overflow was frequency-dependent: at 1-4 Hz, the 3H-overflows were the same; at 8 and 16 Hz, they increased. Cocaine (3 x 10(-5) M) plus corticosterone (4 x 10(-5) M) enhanced the stimulation-evoked 3H-overflow at 1-8 Hz, while it had no effect at 16 Hz. Propranolol (3 x 10(-7) M) did not antagonize this enhancement. An increase in number of pulses from 10 to 1000 in the stimulus caused a corresponding rise in the evoked 3H-overflow. This was also the case when cocaine plus corticosterone were present.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the Mechanism of Neuropeptide Y Induced Potentiation of Neurogenic Vasoconstriction in the Isolated Rabbit Ear Artery

Neuropeptide Y (NPY) has been shown to potentiate the response of the isolated rabbit ear artery to field stimulation, provided that the vascular endothelium is intact. This report describes efforts to elucidate the mechanism by which NPY produces this effect. The response of the perfused rabbit ear artery to brief intermittent field stimulation was significantly enhanced by NPY (100 nmol/L). The administration of indomethacin (10 mumol/L) to inhibit cyclooxygenase did not affect the ability of NPY to potentiate neurogenic vasoconstriction. Blockade of calcium channels with nifedipine (1 mumol/L) produced a partial attenuation of the NPY effect. Our studies therefore suggest that an arachidonic acid metabolite is not involved in NPY induced potentiation of vascular neuroeffector transmission in the rabbit ear artery. On the other hand, the translocation of extracellular calcium may play a role in this process.

Characteristics of chloride currents activated by noradrenaline in rabbit ear artery cells.

1. Responses to noradrenaline were studied in isolated rabbit ear artery cells with the nystatin method of whole-cell patch-clamp recording. With this technique it was possible to obtain reproducible responses to noradrenaline which was not possible with traditional whole-cell recording. 2. With NaCl as the major constituent of the bathing solution (potassium-free pipette and external solutions) the reversal potential (Er) of the noradrenaline-evoked current was about 0 mV. When external chloride was replaced by thiocyanate, iodide, nitrate and bromide, Er was shifted to more negative potentials which indicates that a chloride conductance increase contributes to the current activated by noradrenaline. 3. When sodium was substituted by Tris, N-methyl-D-glucamine, choline or barium, Er of the noradrenaline-evoked current did not alter. This result suggests that a cation conductance is not implicated in the response to noradrenaline recorded with the nystatin method of whole-cell recording. 4. The chloride current activated by noradrenaline was blocked by the selective alpha 1-adrenoceptor antagonist prazosin but was not affected by the alpha 2-adrenoceptor antagonist yohimbine. 5. When cells were exposed to zero calcium bathing solutions the amplitude of the current elicited by noradrenaline was unaffected when measured within 1-2 min in zero calcium conditions. Continued exposure to 0 Ca + 1 mM-EGTA solution reversibly abolished the chloride current to noradrenaline. 6. In the presence of caffeine, which releases Ca2+ from internal stores and itself induced an inward current (at a holding potential of -50 mV), noradrenaline did not elicit a current. These data suggest that the chloride current evoked by noradrenaline results from an increase in intracellular concentration of calcium derived from internal stores. 7. The chloride channel blocking agents 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS; 0.5 mM) and furosemide (0.5 mM) produced partial reduction of the noradrenaline-evoked chloride current whereas 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS), anthracene-9-carboxylic acid (A-9-C) and picrotoxin were ineffective in concentrations of up to 0.5 mM. However DIDS and furosemide were non-selective blockers as both agents were more effective against the adenosine triphosphate-induced cation current.

Involvement of cyclic AMP in the stimulation-evoked noradrenaline release from rabbit isolated ear artery

Involvement of cyclic AMP in the stimulation-evoked noradrenaline release from rabbit isolated ear artery

Distinction of NPY receptors in vitro and in vivo. I. NPY-(18-36) discriminates NPY receptor subtypes in vitro

We studied the possibility of multiple neuropeptide Y (NPY) receptor subtypes. NPY-stimulated Ca2+ mobilization in human erythroleukemia (HEL) cells was used to screen a number of NPY analogues. The potencies of three of these analogues [peptide YY (PYY), [D-Tyr-36]NPY, and NPY-(18-36)] were compared with that of NPY in the following model systems: Ca2+ mobilization and inhibition of adenosine 3',5'-cyclic monophosphate accumulation in HEL cells, potentiation of vasoconstriction in the isolated rabbit ear artery, reduction of cutaneous microvascular perfusion in the rat digit, and inhibition of [3H]serotonin release in rat brain. In each of the five models, PYY was a full agonist that exhibited a similar or slightly higher potency than NPY, whereas [D-Tyr-36]NPY and NPY-(18-36) were partial agonists with lower potencies: NPY-(18-36) had a lower potency and efficacy than [D-Tyr-36]NPY in HEL cells and the rabbit ear artery, but was more effective than [D-Tyr-36]NPY for constricting cutaneous microvasculature and inhibiting serotonin release. Because of its weak partial agonism, we also tested NPY-(18-36) as an antagonist of NPY-stimulated Ca2+ mobilization in HEL cells. NPY-(18-36) shifted the NPY concentration-response curve to the right with a KB affinity value of 297 nM. In summary, [D-Tyr-36]NPY and NPY-(18-36) are partial agonists, the relative potency of which varies between systems. These data demonstrate the presence of multiple NPY receptor subtypes. We propose a modified classification scheme of NPY receptor subtypes.

Inhibition of neurotransmission by angiotensin I and II in rabbit isolated ear artery

Angiotensin I and II inhibited the nerve stimulation-induced pressure changes in isolated, perfused rabbit ear atery with an IC 50 of 3.07 and 0.36 nM, respectively. Neither angiotensin I nor angiotensin II affected the basal pressure or the pressure changes elicited by exogenously administered norepinephrine (NE). The potency of angiotensin I was unaltered by 10 −5 M captopril, indicating that conversion by angiotensin converting enzyme (ACE) was not necessary and did not take place. [Sar 1, Val 5, Ala 8]angiotensin II (3 × 10 −8 M) antagonized the effect of angiotensin I. These findings could have implications regarding ACE inhibitor therapy and the pathophysiology of migraine.

MECHANISMS OF THE EFFECTS OF ENDOTHELIN ON RESPONSES TO NORADRENALINE AND SYMPATHETIC NERVE STIMULATION

1. The effects of endothelin (0.1-1 nmol/L) and the phorbol ester, phorbol 12-myristate 13-acetate (PMA, 0.1-100 nmol/L) have been studied on vasoconstrictor responses to sympathetic nerve stimulation and noradrenaline in the rabbit isolated ear artery. 2. Endothelin (0.1 nmol/L) significantly enhanced the biphasic response of the arteries to a prolonged period of sympathetic nerve stimulation (2 Hz for 60 s). Both the component of the response attributable to mobilization of intracellular calcium (phasic response) and the component due to the influx of extracellular calcium (tonic response) were enhanced. 3. PMA (0.1 and 0.3 nmol/L), which activates protein kinase C, enhanced vasoconstrictor responses to noradrenaline but a higher concentration of PMA (100 nmol/L) inhibited responses to noradrenaline. 4. It is concluded that the enhancement of responses to sympathetic nerve stimulation and noradrenaline produced by endothelin may be due to facilitation of the influx of extracellular calcium and to the activation of protein kinase C.