George J. Baldo

Ion, water and neutral solute transport in Xenopus oocytes expressing frog lens MIP

We have expressed frog (Rana pipiens) lens major intrinsic protein (MIP) in Xenopus oocytes and observed its effect on ion conductance, water permeability and neutral solute transport. SDS-PAGE and immunoblotting demonstrated oocytes injected with MIP mRNA expressed the protein at high levels. Immunolocalization indicated the expressed MIP migrated to the plasma membrane. MIP had no effect on the slope of oocyte I-V relations in the range -50 to +10 mV, although the averaged I-V curve was shifted 10 mV positive to control. MIP increased oocyte water permeability by a factor of 1.9 +/- 0.2, whereas the permeability to sucrose, 2-deoxyglucose, inositol, sorbitol, reduced glutathione or urea was unchanged. Glycerol permeability was enhanced in oocytes expressing MIP. In contrast to control oocytes, 3H-glycerol radioactivity accumulation did not follow first order kinetics. Radioactivity continued to accumulate even after 19 h of uptake and went beyond equilibrium with the bath. The time course of MIP-mediated glycerol uptake was modeled assuming metabolic trapping with good results. Based on this model, MIP increased oocyte glycerol permeability by a factor of 2.7.

Isoprenaline, Ca2+ and the Na(+)-K+ pump in guinea-pig ventricular myocytes.

1. The whole‐cell patch clamp technique was employed to study the effects of the beta‐agonist isoprenaline (ISO) on the Na(+)=K+ pump current, Ip, in acutely isolated ventricular myocytes from guinea‐pig hearts. Propranolol, a beta‐adrenergic antagonist, was used to demonstrate that all of the effects of ISO, stimulatory or inhibitory, are mediated by beta‐receptors. 2. Below about 150 nM [Ca2+]i, we find that ISO reduces Ip, while above this [Ca2+]i ISO increases Ip. The stimulatory and inhibitory effects of ISO on Ip are independent of either intracellular sodium ([Na+]i) or extracellular potassium ([K+]o). These results suggest that the end‐effect of ISO is directly on the maximum pump turnover rate (Vmax) rather than indirectly through changes in [Na+]i or [K+]o or modulatory effects on Na+ or K+ affinity. 3. The maximum effect of ISO increases Ip by 25% when [Ca2+] is buffered at 1.4 microM. A half‐maximal effect is reached at roughly 10 nM‐ISO and a near‐maximal effect by 0.5 microM. 4. The permeabilized patch technique, using amphotericin B (Horn & Marty, 1988; Rae, Cooper, Gates & Watsky, 1991), was employed to minimize changes in the normal second messenger systems and calcium buffers. In these experiments, we used a high intracellular sodium solution (pipette sodium was 50 mM), thus sodium‐calcium exchange was depressed and we expected [Ca2+]i to be above 150 nM. ISO increases Ip in these conditions as in the dialysed cells. 5. Our results suggest that beta‐stimulation can increase Ip, but only if [Ca2+]i is above about 150 nM. In the beating heart [Ca2+]i rises well above this value during systole and the average [Ca2+]i, which depends on heart rate, is expected to normally be above this level. During beta‐stimulation, the increase in Ip along with a concomitant increase in IK (Giles, Nakajima, Ono & Shibata, 1989; Duchatelle‐Gourdon, Hartzell & Lagrutta, 1989) helps prevent action potential lengthening and allows an increase in heart rate even in the presence of increased calcium current. Further, beta‐stimulation will compensate for the effects on Ip of either hypokalaemia or digitalis toxicity, and so reduce the expected rise in both [Na+]i and [Ca2+]i.

α‐Adrenergic effects on Na+‐K+ pump current in guinea‐pig ventricular myocytes

1 The whole‐cell patch clamp was employed to study Na+‐K+ pump current (Ip) in acutely isolated myocytes. α‐Adrenergic receptors were activated with noradrenaline (NA) after blocking β‐adrenergic receptors with propranolol. Ip was measured as the current blocked by strophanthidin (Str). 2 Activation of α‐receptors by NA increased Ip in a concentration‐dependent manner. The K0.5 depended on intracellular calcium ([Ca2+]i), however maximal stimulation did not. At 15 nm[Ca2+]i the K0.5 was 219 nm NA whereas at 1.4 μm [Ca2+]i it was 3 nm. 3 The voltage dependence of Ip was not shifted by NA at either high or low [Ca2+]i. At each voltage, maximal stimulation of Ip was 14‐15 %. 4 Staurosporine (St), an inhibitor of protein kinase C (PKC), eliminated the α‐receptor‐mediated stimulation of Ip at either high or low[Ca2+]i. 5 The stimulation of Ip was independent of changes in intracellular sodium or external potassium concentrations, and did not reflect a change in affinity for Str. 6 Phenylephrine, methoxamine and metaraminol, three selective α1‐adrenergic agonists, stimulate Ip in a similar manner to NA. Stimulation of Ip by NA was eliminated by prazosin, an α1‐antagonist, but was unaffected by yohimbine, an α2‐antagonist. 7 We conclude noradrenaline activates ventricular α1‐receptors, which are specifically coupled via PKC to increase Na+‐K+ pump current. The sensitivity of the coupling is [Ca2+]i dependent, however the maximal increase in pump current is [Ca2+]i and voltage independent.

Regulation of the beta‐stimulation of the Na(+)‐K+ pump current in guinea‐pig ventricular myocytes by a cAMP‐dependent PKA pathway.

1. The whole‐cell patch‐clamp technique was employed with the free intracellular [Ca2+] fixed at 1.4 microM in order to study the isoprenaline (Iso)‐induced increase in the Na(+)‐K+ pump current (Ip) in acutely isolated guinea‐pig ventricular myocytes. 2. The non‐specific protein kinase inhibitor, H‐7, eliminated the stimulatory effect of Iso, suggesting a phosphorylation step is involved in the beta‐agonist stimulation of Ip. 3. H‐7 or the phosphatase inhibitor calyculin A individually had no effect on basal Ip; however, when Ip was first increased by Iso, H‐7 inhibited and calyculin A further increased Ip. This suggests phosphorylation is not important to the basal regulation of Ip, but does have an effect during beta‐stimulation. 4. The Iso‐induced increase in Ip could be mimicked by adding the membrane‐permanent cAMP analogue chlorophenylthio‐cAMP, blocking cAMP degradation with IBMX or stimulating cAMP production with forskolin. Alternatively the protein kinase A inhibitor PKI blocked the stimulatory effect of Iso. This suggests the Iso‐induced phosphorylation responsible for increasing Ip is mediated by cAMP, which then activates protein kinase A (PKA). 5. We conclude that the beta‐agonist‐induced increase in Ip in the presence of high intracellular [Ca2+] is mediated by a phosphorylation step via the cAMP‐dependent PKA pathway. During beta‐stimulation, this increase in active Na(+)‐K+ transport can serve to offset the effects of increases in passive membrane conductances.

THE EFFECTS OF BETA -STIMULATION ON THE NA+-K+ PUMP CURRENT-VOLTAGE RELATIONSHIP IN GUINEA-PIG VENTRICULAR MYOCYTES

1. The whole cell patch clamp technique was used to study effects of the beta agonist isoprenaline (Iso) on the current‐voltage (I‐V) relationship of the Na(+)‐K+ pump current (Ip) in acutely isolated guinea‐pig ventricular myocytes. 2. The effect of Iso on Ip at high [Ca2+]i (1.4 microM) was voltage dependent. The I‐V relationship of Ip in Iso shifted by approximately 30 mV in the negative direction on the voltage axis, increasing Ip at negative voltages but leaving Ip unchanged at positive voltages. 3. Intracellular application of the calmodulin antagonist, calmodulin‐dependent protein kinase II fragment 290‐309, did not eliminate or reduce the Iso‐induced voltage shift, suggesting calmodulin‐dependent protein kinase II was not involved. 4. The Iso inhibition of Ip at low [Ca2+]i (15 nM) was not voltage dependent. Ip was reduced by 20 to 30% in the presence of Iso at each holding potential. 5. When the voltage dependence of Ip was largely reduced by substitution of N‐methyl‐D‐glucamine+ for external Na+, the magnitude of the low [Ca2+]i, Iso‐induced inhibition of Ip was progressively eliminated by increasing the [Ca2+]i. At a [Ca2+]i of 1.4 microM, this inhibition disappeared. 6. At intermediate values of [Ca2+]i, the I‐V curves in Na(+)‐containing solution in the presence and the absence of Iso crossed over. The higher the [Ca2+]i, the more positive the voltage at which the two I‐V curves intersected. 7. During beta‐adrenergic activation our results suggest intracellular Ca2+ has two effects: (a) It prevents protein kinase A (PKA) phosphorylation‐induced inhibition of Ip. (b) It causes a PKA phosphorylation‐induced shift of the pump I‐V relationship in the negative direction on the voltage axis. These effects may have important physiological significance in the regulation of heart rate and cardiac contractility.

Activation of PKC increases Na+-K+ pump current in ventricular myocytes from guinea pig heart

Abstract We have previously shown activation of α1-adrenergic receptors increases Na+-K+ pump current (Ip) in guinea pig ventricular myocytes, and the increase is eliminated by blockers of phosphokinase C (PKC). In this study we examined the effect of activators of PKC on Ip. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased IP at each test potential without shifting its voltage dependence. The concentration required for a half-maximal response (K0.5) was 6 µM at 15 nM cytosolic [Ca2+] ([Ca2+]i) and13 nM at 314 nM [Ca2+]i. The maximal increase at either [Ca2+]i was about 30%. Another activator of PKC, 1,2-dioctanoyl-sn-glycerol (diC8), increased Ip similarly. The effect of PMA on IP was eliminated by the PKC inhibitor staurosporine, but not by the peptide PKI, an inhibitor of protein kinase A (PKA). PMA and α1-adrenergic agonist effects both were sensitive to [Ca2+]i, blocked by PKC inhibitors, unaffected by PKA inhibition, and increased Ip uniformly at all voltages. However, they differed in that α1-activation caused a maximum increase of 15% vs 30% via PMA, and α1-effects were less sensitive to [Ca2+]i than PMA effects. These results demonstrate that activation of PKC causes an increase in Ip in guinea pig ventricular myocytes. Moreover, they suggest that the coupling of α1-adrenergic activation to Ip is entirely through PKC, however α1-activation may be coupled to a specific population of PKC whereas PMA is a more global agonist.

Discrete Charges on Biological Membranes

The main objective of this chapter is to derive an exact expression for the potential due to a single charge at an interface. then examine the validity of the limiting Debye-Huckel expression when the dielectric constant of the membrane phase has a finite value characteristic of a biological membrane.

The cDNA sequence encoding the major intrinsic protein of frog lens

A cDNA clone encoding the frog lens major intrinsic protein (MIP) has been isolated and sequenced. The predicted protein of 28 kDa has high sequence identity and similarity to mammalian and avian lens MIP sequences. Frog lens MIP is encoded by a transcript of 4.4 kb.

Effects of acetylcholine on the Na+–K+ pump current in guinea‐pig ventricular myocytes

1 The whole‐cell patch clamp technique was used to study the effects of acetylcholine (ACh) on Na+‐K+ pump current (Ip) in acutely isolated guinea‐pig ventricular myocytes. Studies were performed in the absence and presence of the β‐agonist isoprenaline (Iso). 2 ACh had no effect on Ip at low or high [Ca2+]i at any voltage in the absence of Iso. Iso alone inhibited Ip at low [Ca2+]i and shifted the Ip–V relationship at high [Ca2+]i in a negative direction. Addition of 1 μm ACh reversed these effects of Iso. K0.5 for the effects of ACh was about 16 nm, regardless of [Ca2+]i. 3 The actions of ACh on the heart are usually mediated via muscarinic receptors. Atropine, a muscarinic antagonist, blocked the effects of ACh on Ip in the presence of Iso, suggesting that these effects are also mediated by muscarinic receptors. 4 Muscarinic receptors are usually coupled to a Gi protein, leading to inhibition of adenylyl cyclase and a reduction of cAMP levels. We have shown previously that basal levels of cAMP are very low in guinea‐pig ventricular myocytes, and that a membrane‐permeant cAMP analogue, chlorophenylthio‐cAMP (CPTcAMP), mimics the effects of Iso. ACh did not reverse the effects of CPTcAMP, supporting the hypothesis that the effects of ACh on Ip are also mediated via inhibition of adenylyl cyclase. 5 The present results suggest that a high level of parasympathetic tone alone does not affect the activity of ventricular Na+–K+ pumps. However, if sympathetic tone is high, then muscarinic stimulation can reciprocally modulate Na+–K+ pump activity.

Effects of neuronal bungarotoxin and nitric oxide inhibitors on the post-iontophoretic burst of miniature end-plate currents at the frog neuromuscular junction

INTRODUCTION In an earlier report, we described a phenomenon in which the application of acetylcholine (ACh) to frog motor end-plates for 10-30 s produced a transient elevation in miniature end-plate current (m.e.p.c.) frequency (1). This burst of m.e.p.c.s is an approximate 4-5 fold increase over background frequency, lasting 20-30 s and beginning only near the end of the ACh application (either iontophoretic or pressureejected), even when the duration of the ACh pulse is extended from 10 to 30 s. Usually the burst decreased in intensity over the course of repeated ACh pulses; once the burst had disappeared it usually could not be restored by rest periods lasting up to several hours. Experiments with ion substitutions, drugs and channel blockers indicated: 1) Na + did not affect the burst; 2) Ca z+ was required but could be replaced by Sr2+; 3) ACh analogs, including carbachol, acetyl-[3methylcholine and the pure nicotinic agonist 1,1dimethyl-4-phenyl-piperazinium iodide (DMPP) produced the burst; 4) presynaptic muscarinic receptors were not obviously involved. We would have attributed these phenomena to presynaptic nicotinic receptors (see 2 for review), but were forced to reject this explanation because of a correlation between muscle membrane potential during the ACh pulse and the resulting burst intensity: voltage-clamping the end-plate at -100mV and stepping to -60mV during the ACh