18P PROCEEDINGS OF THE (1968). They are secured in specially designed micro-electrode holders which are attached to Prior micromanipulators. An electrode is normally driven into a quiescent fibre although penetration of the thick external collagenous sheath can be facilitated by eliciting a single contraction while the microelectrode is pressed against the fibre's surface. The apparatus has been designed to make it possible to voltage clamp and examine contractility concurrently. Gibbons & Fozzard (1971) using two micro-electrodes and a photoelectric device for measuring tension have shown the feasibility and value of undertaking such simultaneous measurements in the Purkinje fibre. In the light of recently published work on the action of low doses of ouabain on Purkinje fibre contractility (Blood, 1975) and ionic currents (Cohen, Daut & Noble, 1976) the correlation of these results can now be examined. REFERENCES
BLOOD, B. E. (1975). J. Physiol. 251, 69-70P. BLOOD, B. E. & SPINDLER, A. J. (1975). J. Physiol. 252, 2-3P. COHEN, I., DAUT, J. & NOBLE, D. (1976). J. Physiol. 260, 55-74. GIBBONS, W. R. & FOZZARD, H. A. (1971). Circulation Res. 28, 446-460. TASAKI, K., TSUKAHAERA, Y., ITO, S., WAYNER, M. J. & YU, W. Y. (1968). Physiol. & Behav. 3, 1009-1110.
COMMUNICATIONS Induction of the acrosome reaction in guinea-pig spermatozoa in vitro by the Ca ionophore A23187 BY D. P. L. GREEN. Physiological Laboratory, Downing Street, Cambridge CB2 3EG Mammalian spermatozoa possess an intracellular organelle, the acrosome, which overlies the nucleus and whose discharge, known as the acrosome reaction, occurs in close proximity to the ovum. The acrosome reaction is a necessary condition for penetration by spermatozoa of the zona pellucida surrounding the ovum, and subsequent gamete fusion. Both ejaculated and epididymal spermatozoa require a period of capacitation, a process which normally occurs in the female tract but which may be produced in vitro in certain species. In the guinea-pig, capacitation and the acrosome reaction occur in 40-60 % of spermatozoa after incubation in a simple medium for 10-15 hr. Recently, Yanagimachi & Usui (1974) have established the dependence of the acrosome reaction under these circumstances on the external calcium concentration. In order to assess whether an increase in the intracellular calcium concentration is an important part *
M.R.C. scholar.
19P PHYSIOLOGICAL SOCIETY, MA Y 1976 of the acrosome reaction, the effect of the Ca ionophore A23187 on guineapig spermatozoa was studied. A medium containing 140 mM-NaCl, 4 mm-KCl, 4 mm Hepes, 1O mM glucose and either 2 mM-CaCl2 or 2 MM-MgCl2, pH7-4, wasused throughout and spermatozoa were inspected by dark field and phase-contrast light microscopy for the acrosome reaction. In calcium medium, addition of A23187, final concentration 4/uM, in dimethyl sulphoxide induced the acrosome reaction in more than 95 % of intact spermatozoa within 10 min at 370 C whereas the same medium, with or without dimethyl sulphoxide, but with no A23187, showed less than 5 % with the acrosome reaction within the same period. In magnesium medium, addition of A23187 showed no difference from the controls. The extent of the acrosome reaction was estimated independently by enzymic determination of proteinase activity released by acrosomal discharge. Electron micrographs confirm the observations made by light microscopy and demonstrate the morphological normality of the acrosome reaction induced by A23187. These results suggest that an increase in the intracellular calcium concentration is a crucial step in inducing the acrosome reaction. The stimulus for the acrosome reaction in vivo is not known but, if calcium entry is a normal consequence of the stimulus, it appears not to be through a potential dependent calcium channel since replacement of sodium by isotonic potassium, in medium containing calcium but no ionophore, was ineffective in inducing the acrosome reaction over a period of 1 hr. There is a clear similarity between the acrosome reaction of spermatozoa and the exocytosis of the contents of intracellular organelles in secretary cells, a process which appears to be dependent, in most cases, upon a rise in the intracellular calcium concentration. A23187 was a gift of Eli Lilley and Co, Indianapolis. REFERENCE
YANAGIMAC:EII, R. & Usui, N. (1974). Expl Cell Res. 89, 161-174.
The action of some vasoactive polypeptides and their antagonists on the anococcygeus muscle BY J. S. GILLESPIE and A. T. MCKNIGHT. Department of Pharmacology, University of Glasgow, Glasgow G12 8QQ Studies on anococcygeus muscles from several species have shown that while their receptor populations differ, all have in common a motor innervation which is adrenergic and an inhibitory innervation whose transmitter is unknown. Stimulation of the inhibitory nerves in vivo caused inhibition of the anococcygeus and a fall in blood pressure suggesting
20P PROCEEDINGS OF THE the transmitter had vasodilator properties. We have, therefore, examined the effects of three biologically occurring vasodilator polypeptides bradykinin, substance P and eledoisin - on the anococcygeus in vitro to see whether they possessed the properties required of an inhibitory transmitter. In particular we have tested whether the polypeptides could produce inhibition, and if so whether this inhibition summated with the inhibitory response to nerve stimulation; and finally whether antagonists of the polypeptides also antagonize responses to inhibitory nerve stimulation. In the untreated rat anococcygeus, lacking intrinsic tone, bradykinin (10-8 to 104 g/ml.) had either no effect or produced a small, transient rise in tone; substance P (10-7 to 10-5 gml.) had no effect, but at higher concentrations reduced responses to motor nerve stimulation; eledoisin (10-7 to 104 g/ml.) consistently contracted the muscle and reduced responses to motor nerve stimulation. The eledoisin contractions were slow to develop and completely abolished by phentolamine (106 M) suggesting they were due to release of noradrenaline from adrenergic nerves. In the contracted rat anococcygeus inhibitory nerve stimulation, bradykinin and substance P, but not eledoisin, inhibited guanethidine induced tone. The inhibitory responses to substance P were seen only with concentrations higher than 106 gfml. and were small and slow to develop. Bradykinin (10-8 to 104M) in contrast produced a rapid and large inhibitory response comparable to the response to inhibitory nerve stimulation in preparations from rat, rabbit and cat. None of the polypeptides markedly affected the magnitude of responses to inhibitory nerve stimulation. These results suggested that of the three, only bradykinin could be the inhibitory transmitter, therefore we examined the effects of two bradykinin antagonists, hesperetin and khellin, on bradykinin inhibition and the responses to inhibitory nerve stimulation in the anococcygeus. Neither hesperetin nor khellin (10-6 to 104 M) antagonized the inhibitory effects of bradykinin or of nerve stimulation; indeed both were on occasions potentiated, particularly the response to nerve stimulation at low frequency. This may be related to the ability of both hesperetin and khellin themselves to produce inhibition in both rat and rabbit anococcygeus. In summary, we conclude that bradykinin, or a substance related to it, may have the properties required of an inhibitory transmitter in the
anococcygeus.
PH YSIOLOGICAL SOCIETY, MA Y 1976
21P
The use of sodium salicylate to counter glycoside toxicity BY I. COHEN, D. NOBLE and C. OJEDA. University Laboratory of Physiology, Oxford OX1 3PT Cohen, Daut & Noble (1975) studied Purkinje fibre membrane currents in the presence of ouabain. The toxicity of a given dose was critically dependent on [K]O. We have since found that similar toxic effects of acidity and high [Ca]. are countered by increasing [K]0 to 108 mm. However, in the whole animal, [K]o > 6 mm produces serious arrhythmias. We therefore decided to see whether protection from toxicity may depend on local [K] near the membrane rather than on [K]O. Sodium salicylate was used to increase membrane surface negative charge (McLaughlin, 1973). Ten mm salicylate completely prevented the marked shortening of ventricular action potentials induced by a toxic dose (10-6 M) of ouabain. Instead, action potential duration increased. A toxic action may also be reversed by adding salicylate after ouabain (Fig. 1). Again, the duration is increased above the control value. (Salicylate alone is without marked effect on duration.) At a later stage the muscle becomes inexcitable due to an increase in threshold which also occurs in salicylate alone and might be attributed to a change in surface charge. Control
10 min 10'6Mouabain
>
BLOOD, B. E. (1975). J. Physiol. 251, 69-70P. BLOOD, B. E. & SPINDLER, A. J. (1975). J. Physiol. 252, 2-3P. COHEN, I., DAUT, J. & NOBLE, D. (1976). J. Physiol. 260, 55-74. GIBBONS, W. R. & FOZZARD, H. A. (1971). Circulation Res. 28, 446-460. TASAKI, K., TSUKAHAERA, Y., ITO, S., WAYNER, M. J. & YU, W. Y. (1968). Physiol. & Behav. 3, 1009-1110.
COMMUNICATIONS Induction of the acrosome reaction in guinea-pig spermatozoa in vitro by the Ca ionophore A23187 BY D. P. L. GREEN. Physiological Laboratory, Downing Street, Cambridge CB2 3EG Mammalian spermatozoa possess an intracellular organelle, the acrosome, which overlies the nucleus and whose discharge, known as the acrosome reaction, occurs in close proximity to the ovum. The acrosome reaction is a necessary condition for penetration by spermatozoa of the zona pellucida surrounding the ovum, and subsequent gamete fusion. Both ejaculated and epididymal spermatozoa require a period of capacitation, a process which normally occurs in the female tract but which may be produced in vitro in certain species. In the guinea-pig, capacitation and the acrosome reaction occur in 40-60 % of spermatozoa after incubation in a simple medium for 10-15 hr. Recently, Yanagimachi & Usui (1974) have established the dependence of the acrosome reaction under these circumstances on the external calcium concentration. In order to assess whether an increase in the intracellular calcium concentration is an important part *
M.R.C. scholar.
19P PHYSIOLOGICAL SOCIETY, MA Y 1976 of the acrosome reaction, the effect of the Ca ionophore A23187 on guineapig spermatozoa was studied. A medium containing 140 mM-NaCl, 4 mm-KCl, 4 mm Hepes, 1O mM glucose and either 2 mM-CaCl2 or 2 MM-MgCl2, pH7-4, wasused throughout and spermatozoa were inspected by dark field and phase-contrast light microscopy for the acrosome reaction. In calcium medium, addition of A23187, final concentration 4/uM, in dimethyl sulphoxide induced the acrosome reaction in more than 95 % of intact spermatozoa within 10 min at 370 C whereas the same medium, with or without dimethyl sulphoxide, but with no A23187, showed less than 5 % with the acrosome reaction within the same period. In magnesium medium, addition of A23187 showed no difference from the controls. The extent of the acrosome reaction was estimated independently by enzymic determination of proteinase activity released by acrosomal discharge. Electron micrographs confirm the observations made by light microscopy and demonstrate the morphological normality of the acrosome reaction induced by A23187. These results suggest that an increase in the intracellular calcium concentration is a crucial step in inducing the acrosome reaction. The stimulus for the acrosome reaction in vivo is not known but, if calcium entry is a normal consequence of the stimulus, it appears not to be through a potential dependent calcium channel since replacement of sodium by isotonic potassium, in medium containing calcium but no ionophore, was ineffective in inducing the acrosome reaction over a period of 1 hr. There is a clear similarity between the acrosome reaction of spermatozoa and the exocytosis of the contents of intracellular organelles in secretary cells, a process which appears to be dependent, in most cases, upon a rise in the intracellular calcium concentration. A23187 was a gift of Eli Lilley and Co, Indianapolis. REFERENCE
YANAGIMAC:EII, R. & Usui, N. (1974). Expl Cell Res. 89, 161-174.
The action of some vasoactive polypeptides and their antagonists on the anococcygeus muscle BY J. S. GILLESPIE and A. T. MCKNIGHT. Department of Pharmacology, University of Glasgow, Glasgow G12 8QQ Studies on anococcygeus muscles from several species have shown that while their receptor populations differ, all have in common a motor innervation which is adrenergic and an inhibitory innervation whose transmitter is unknown. Stimulation of the inhibitory nerves in vivo caused inhibition of the anococcygeus and a fall in blood pressure suggesting
20P PROCEEDINGS OF THE the transmitter had vasodilator properties. We have, therefore, examined the effects of three biologically occurring vasodilator polypeptides bradykinin, substance P and eledoisin - on the anococcygeus in vitro to see whether they possessed the properties required of an inhibitory transmitter. In particular we have tested whether the polypeptides could produce inhibition, and if so whether this inhibition summated with the inhibitory response to nerve stimulation; and finally whether antagonists of the polypeptides also antagonize responses to inhibitory nerve stimulation. In the untreated rat anococcygeus, lacking intrinsic tone, bradykinin (10-8 to 104 g/ml.) had either no effect or produced a small, transient rise in tone; substance P (10-7 to 10-5 gml.) had no effect, but at higher concentrations reduced responses to motor nerve stimulation; eledoisin (10-7 to 104 g/ml.) consistently contracted the muscle and reduced responses to motor nerve stimulation. The eledoisin contractions were slow to develop and completely abolished by phentolamine (106 M) suggesting they were due to release of noradrenaline from adrenergic nerves. In the contracted rat anococcygeus inhibitory nerve stimulation, bradykinin and substance P, but not eledoisin, inhibited guanethidine induced tone. The inhibitory responses to substance P were seen only with concentrations higher than 106 gfml. and were small and slow to develop. Bradykinin (10-8 to 104M) in contrast produced a rapid and large inhibitory response comparable to the response to inhibitory nerve stimulation in preparations from rat, rabbit and cat. None of the polypeptides markedly affected the magnitude of responses to inhibitory nerve stimulation. These results suggested that of the three, only bradykinin could be the inhibitory transmitter, therefore we examined the effects of two bradykinin antagonists, hesperetin and khellin, on bradykinin inhibition and the responses to inhibitory nerve stimulation in the anococcygeus. Neither hesperetin nor khellin (10-6 to 104 M) antagonized the inhibitory effects of bradykinin or of nerve stimulation; indeed both were on occasions potentiated, particularly the response to nerve stimulation at low frequency. This may be related to the ability of both hesperetin and khellin themselves to produce inhibition in both rat and rabbit anococcygeus. In summary, we conclude that bradykinin, or a substance related to it, may have the properties required of an inhibitory transmitter in the
anococcygeus.
PH YSIOLOGICAL SOCIETY, MA Y 1976
21P
The use of sodium salicylate to counter glycoside toxicity BY I. COHEN, D. NOBLE and C. OJEDA. University Laboratory of Physiology, Oxford OX1 3PT Cohen, Daut & Noble (1975) studied Purkinje fibre membrane currents in the presence of ouabain. The toxicity of a given dose was critically dependent on [K]O. We have since found that similar toxic effects of acidity and high [Ca]. are countered by increasing [K]0 to 108 mm. However, in the whole animal, [K]o > 6 mm produces serious arrhythmias. We therefore decided to see whether protection from toxicity may depend on local [K] near the membrane rather than on [K]O. Sodium salicylate was used to increase membrane surface negative charge (McLaughlin, 1973). Ten mm salicylate completely prevented the marked shortening of ventricular action potentials induced by a toxic dose (10-6 M) of ouabain. Instead, action potential duration increased. A toxic action may also be reversed by adding salicylate after ouabain (Fig. 1). Again, the duration is increased above the control value. (Salicylate alone is without marked effect on duration.) At a later stage the muscle becomes inexcitable due to an increase in threshold which also occurs in salicylate alone and might be attributed to a change in surface charge. Control
10 min 10'6Mouabain
>
