Confocal ratio-imaging of intracellular pH in unfertilised mouse oocytes.

Unfertilised mouse oocytes absorbed the pH-sensitive fluoroprobe SNARF-1-AM (carboxyseminaph-thorhodafluor-1-acetoxymethylester), the ester being hydrolysed by an intracellular esterase. Ratio-imaging of oocytes containing the resultant SNARF-1 excited by laser light (514 nm) has been obtained by scanning confocal microscopy with appropriate barrier filters to monitor emission maxima about 590 and 640 nm recorded simultaneously in separate channels of the framestore. Images produced by pixel-by-pixel division of these channel images showed uniform distribution of SNARF-1 in equatorial regions in most cells. However, in some oocytes regions (about 4 microns diameter) with smaller ratios (i.e. lower pHi) were detected. The relation between the ratio of emitted maxima and the extracellular pH (pHo) in the presence of nigericin allowed a calibration procedure to determine the intracellular pH (pHi). With this method pHi was estimated to be 7.13 +/- 0.05 (mean +/- SEM, n = 31). Whereas the application of a weak acid (butyric) caused a fall in the ratio and hence in pHi, exposure to weak bases (NH4Cl or trimethylamine) caused a rise. Large changes in pHo did not evoke corresponding changes in the ratio and hence in pHi. Addition of 5% CO2 to the external solution buffered at the usual value of pH 7.4, however, did cause a fall in the ratio which was reversible only when HCO3- was present in the external solution.

Conductance rise induced by the calcium ionophore A23187 in unfertilized eggs of tilapia.

Current-clamp measurements on unfertilized tilapia eggs gave linear current-voltage relations in the range 0 to -120 mV. Eggs had low resting potentials (-22 mV) and high specific membrane resistances (500 k omega cm2) and specific membrane capacitances (0.9 microF cm-2) indicating no marked folding of their plasma membrane. Application of A23187 induced a small depolarization and a conductance rise, the response having a reversal potential of about -16 mV. The results suggest that tilapia eggs have a calcium-gated conductance probably activated at fertilization.

Single Ca2(+)-activated K+ channels in excised membrane patches of hamster oocytes.

The properties of the Ca2(+)-activated K+ channel in unfertilized hamster oocytes were investigated at the single-channel level using inside-out excised membrane patches. The results indicate a new type of Ca2(+)-activated K+ channel which has the following characteristics: (1) single-channel conductance of 40-85 pS for outward currents in symmetrical K+ (150 mM) solutions. (2) inward currents of smaller conductance (10-50 pS) than outward currents, i.e. the channel is outwardly rectified in symmetrical K+ solutions, (3) channel activity dependent on the internal concentration of free Ca2+ and the membrane potential, (4) modification of the channel activity by internal adenosine 5' diphosphate (0.1 mM) producing a high open probability regardless of membrane potential.

Functional expression of rat pituitary gonadotrophin-releasing hormone receptors in Xenopus oocytes.

Expression of receptors for the hypothalamic regulatory peptide, gonadotrophin-releasing hormone (GnRH), was investigated by intracellular recording from Xenopus oocytes injected with poly(A)+ mRNA isolated from rat anterior pituitary glands. Membrane depolarizations were induced in oocytes in a dose-dependent fashion following the application of GnRH (10nM - 1 microM) or a GnRH superactive agonist, buserelin (1nM - 1 microM). The response was reversibly blocked by the addition of a GnRH antagonist (1 microM). TRH (10nM - 1 microM) had no effect on most of these oocytes. In contrast, some other oocytes which showed no responses to GnRH or to the GnRH agonist, displayed depolarizing responses to TRH (10nM - 1 microM). A relatively small number of oocytes responded to both ligands. Control oocytes did not respond to the GnRH analogues or to TRH. This successful expression of the GnRH receptor could provide a new approach to the study of the receptor, and serve as a means for the isolation and cloning of the encoding genes.

A delayed all-or-none hyperpolarisation induced by a single Ca action potential in hamster eggs.

A transient change of membrane potential and resistance could be evoked after a long latency (ca. 9 s) by a single calcium action potential in some unfertilized hamster eggs. The estimated reversal potential for the delayed response was close to EK supporting the conclusion that K channels were opened indirectly by the Ca2+ influx through voltage-gated channels. A second action potential elicited after the first did not induce a similar response. A number of treatments (insertion of a Ca2+ pipette, application of Na+-free solution, La3+ or high external pH) likely to raise [Ca2+]i also induced similar large changes of potential and resistance after which a single action potential failed to evoke a large delayed response. The evidence indicates that a small rise in [Ca2+]i activates a slow process leading to a further large increase in [Ca2+]i.

On the mechanism of a pH-induced rise in membrane potassium conductance in hamster eggs.

1. The effect of external pH (pHo) on the membrane potential and resistance of unfertilized zona-free hamster eggs was investigated by intracellular recording techniques. 2. A hyperpolarization of the hamster egg membrane was induced by raising the extracellular pH above 8.0. This hyperpolarization was accompanied by a rise in membrane conductance and was reversible by washing the egg. 3. The estimated value of the reversal potential of the hyperpolarizing response to a solution with pHo 9.5 was about -85 mV. The membrane potential changed linearly with log [K+]o with a slope of 43 +/- 2 mV (mean +/- S.D.; n = 4) for a 10-fold change in [K+]o, while it was unaltered by the removal of Cl- from the solution. 4. The amplitude of the pHo-induced hyperpolarization decreased substantially as [Ca2+]o was lowered from 20 to 1 mM. Sr2+ could substitute for Ca2+ in sustaining the response to high pHo, whereas Ba2+ or Mg2+ could not. 5. Injection of the Ca2+ chelator EGTA into the egg prevented the pHo-induced hyperpolarization suggesting that a rise in [Ca2+]i is required. 6. The rate of rise of Ca2+ action potentials was reversibly enhanced by raising pHo. However, influx through the voltage-gated Ca2+ channels is not involved in initiation and maintenance of the pHo-induced response, as responses were not affected by the Ca2+ channel blocker La3+. 7. The duration of the hyperpolarization evoked by intracellular Ca2+ injection in eggs bathed in normal solution or Na+-free solution was greatly prolonged by raising pHo. 8. It is suggested that a rise in external pH produces an increase in [Ca2+]i, activating a Ca2+-mediated K+ conductance which hyperpolarizes the egg membrane. 9. It is concluded that both a Na+-Ca2+ exchange system and a Ca2+ pump are responsible for Ca2+ extrusion and that inhibition of the Ca2+ pump by high pHo is the chief mechanism underlying the pH-induced hyperpolarization in hamster eggs. Although the Na+-Ca2+ exchange system is facilitated at high pHo, the effect of this facilitation of efflux is outweighed by the inhibition of the Ca2+ pump.

The effect of lanthanum, quercetin and dinitrophenol on calcium-evoked electrical responses in hamster eggs.

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.

Intracellular and whole-cell recordings from zona-free hamster eggs: significance of leak impalement artifact.

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.

An electrophysiologic study of rabbit ciliary epithelium.

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.

Calcium action potentials in unfertilized eggs of mice and hamsters.

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.

Cyclic AMP as a possible mediator of dopamine stimulation of cockroach gland cells.

Isolated salivary glands of the cockroach Nauphoeta cinerea Olivier secrete fluid in response to nerve stimulation or application of dopamine, the acinar cells undergoing a hyperpolarization during secretion. The aim of the present work was to examine whether cyclic AMP acts as a second messenger in the acinar cells to cause the secretory and electrical responses to the transmitter dopamine. Cyclic AMP (10-500 microM) in the bathing solution of isolated glands caused a dose-dependent secretory response but no change in the membrane potential of acinar cells. The time courses and magnitudes of the secretory responses to cyclic AMP resembled those features of responses to dopamine. Forskolin, an adenylate cyclase activator, caused fluid secretion but the responses were small and irregular. The phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine (IBMX)(1-1000 microM) produced fluid secretion in a dose-dependent manner, the maximal response being equal to that of dopamine. Maintained responses to cyclic AMP or IBMX required the presence of extracellular calcium ions. An inhibitor (MDL 12,330A) of adenylate cyclase suppressed the secretory responses to dopamine, cyclic AMP, IBMX, the ionophore A23817 or the readmission of calcium ions to the bathing solution; this inhibitor did not block the acinar hyperpolarization caused by nerve stimulation. Cyclic AMP stimulation of glands, bathed in chloride-free solution to prevent fluid secretion, produced a change in the gland cells which outlasted the period of exogenous cyclic AMP stimulation and expressed itself as a transient secretion upon return of the normal bathing solution. It was concluded that stimulus-secretion coupling in this gland involves a calcium-dependent second messenger system and that cyclic AMP is probably the second messenger. The evidence did not support the idea that cyclic AMP is also a second messenger for the acinar cell hyperpolarization evoked by nerve stimulation.

Calcium-evoked opening of potassium channels in hamster eggs.

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.

Chlorotetracycline fluorescence associated with plasma membranes of cockroach salivary gland cells.

The fluorescent compound chlorotetracycline (CTC) enters the cells of the cockroach salivary gland. The acinar peripheral cells and the non-secretory duct cells become preferentially labelled by CTC. Microscopic examination of the intracellular distribution of CTC indicates that this compound labels the highly folded apical plasma membranes of the peripheral cells and the deep infolds of the basal plasma membranes of the non-secretory duct cells. Lanthanum blocks the entry of CTC into all of the gland cells and in this condition the CTC labels the basal surfaces of the acini and ducts. The results of this investigation support the idea that CTC labels calcium ions in the vicinity of plasma membranes.

Uptake of chlorotetracycline into mouse eggs.

The fluorescent compound chlorotetracycline (CTC) enters the mouse egg by a mechanism controlled by the oolemma. The intracellular distribution of CTC is non-uniform and during the early stages of uptake fluorescent patches are observed at the egg's surface probably close to the oolemma. Lanthanum blocks the entry of CTC into the egg's cytoplasm and in this condition the CTC remains at the surface of the egg; the labelled site is not the zona pellucida and is likely to be the outer surface of the oolemma. The results of this study of CTC uptake support the idea that this compound labels divalent (or trivalent) cations in the neighbourhood of cell membranes.

The influence of calcium on the control of fluid secretion in the cockroach salivary gland.

When cockroach salivary glands are bathed in calcium-free medium the basal rate of fluid secretion increases from about 1 nl/min to about 10 nl/min; maintained dopamine stimulation elicits a further rise in secretory rate which gradually declines. Evidence is presented which indicates that magnesium is unable to substitute for calcium in this system. When calcium is returned to the bathing solution after a period of calcium deprivation there is a transient increase in secretory rate. Stimulation of the glands in certain conditions which inhibit the secretory response leads to some kind of calcium-dependent active state in the secretory cells which can outlast the interaction of the agonist with its receptors. It is concluded that stimulus-secretion coupling in this gland involves a calcium-dependent second messenger system.

A calcium-readmission response recorded from Nauphoeta salivary gland acinar cells.

1. When cockroach salivary glands are exposed to bathing solutions without added calcium, reintroduction of calcium causes the acinar cells to hyperpolarize. The effect ('readmission response') may be very prolonged if the conditioning solution contains 5 mM-cobalt. 2. Evidence is presented against the possibility that the readmission response is mediated by transmitter released from the salivary nerves. 3. The readmission response is shown to reflect an increase in potassium conductance.

On the role of calcium in the electrical responses of cockroach salivary gland cells to dopamine.

1. The calcium dependence of the intracellularly recorded hyperpolarizing responses of salivary gland acinar cells of Nauphoeta cinerea (Olivier) to ionophoretically applied dopamine has been examined. The results of withdrawing calcium from the bathing solution were essentially the same whether or not other divalent cations were present. The effects of calcium withdrawal were rapidly reversed on replacement of calcium. 2. Small responses to dopamine were reduced or abolished by calcium withdrawal but could be restored by an increase in the amount of ejected dopamine. Calcium withdrawal did not have any consistent effect on the input resistance: a reduction in input resistance is therefore not the main cause of the reduction in the amplitude of the responses to dopamine. 3. In very low calcium solutions responses elicited by repeated prolonged dopamine applications progressively declined. 4. It is suggested that the hyperpolarizing responses to dopamine depend on an influx of calcium into the cytosol from a store which can be replenished only from the exterior.

alpha-Flupenthixol: an antagonist of dopamine-evoked fluid secretion by an insect salivary gland preparation.

1 We have demonstrated inhibition of secretory responses of cockroach salivary glands to dopamine, adrenaline, noradrenaline and neurotransmitter by alpha-flupenthixol. This inhibition was slow in onset (60 min) and in reversal (greater than 2 h). 2 Inhibition of responses to adrenaline and noradrenaline was non-competitive, since the maxima and slopes of dose-response curves of these agonists were reduced. 3 Although at low concentrations (less than 3 microM) the antagonism of responses to dopamine showed some characteristics of competitive inhibition, at higher doses non-competitive inhibition was clearly demonstrated. 4 These results are explained in terms of different efficacies of the agonists for the receptors antagonized by alpha-flupenthixol. 5 beta-Flupenthixol was shown to antagonize responses to dopamine; however it was 10 to 100 times less potent than alpha-flupenthixol.

A novel effect of cobalt treatment on calcium-dependent responses of the cockroach salivary gland.

After incubation in calcium-free solutions containing cobalt, the readmission of calcium caused prolonged but reversible hyperpolarization of acinar cells of cockroach salivary glands and prolonged fluid secretion. It is suggested that cobalt treatment increases the permeability of the acinar cell membrane to calcium.