Two functionally different Na/K pumps in cardiac ventricular myocytes.

The whole-cell patch-clamp technique was used to voltage clamp acutely isolated myocytes at -60 mV and study effects of ionic environment on Na/K pump activity. In quiescent guinea pig myocytes, normal intracellular Na+ is approximately 6 mM, which gives a total pump current of 0.25 +/- 0.09 pA/pF, and an inward background sodium current of 0.75 +/- 0.26 pA/pF. The average capacitance of a cell is 189 +/- 61 pF. Our main conclusion is the total Na/K pump current comprises currents from two different types of pumps, whose functional responses to the extracellular environment are different. Pump current was reversibly blocked with two affinities by extracellular dihydro-ouabain (DHO). We determined dissociation constants of 72 microM for low affinity (type-1) pumps and 0.75 microM for high affinity (type-h) pumps. These dissociation constants did not detectably change with two intracellular Na+ concentrations, one saturating and one near half-saturating, and with two extracellular K+ concentrations of 4.6 and 1.0 mM. Ion effects on type-h pumps were therefore measured using 5 microM DHO and on total pump current using 1 mM DHO. Extracellular K+ half-maximally activated the type-h pumps at 0.4 mM and the type-1 at 3.7 mM. Extracellular H+ blocked the type-1 pumps with half-maximal blockade at a pH of 7.71 whereas the type-h pumps were insensitive to extracellular pH. Both types of pumps responded similarly to changes in intracellular-Na+, with 9.6 mM causing half-maximal activation. Neither changes in intracellular pH between 6.0 and 7.2, nor concentrations of intracellular K+ of 140 mM or below, had any effect on either type of pump. The lack of any effect of intracellular K+ suggests the dissociation constants are in the molar range so this step in the pump cycle is not rate limiting under normal physiological conditions. Changes in intracellular-Na+ did not affect the half-maximal activation by extracellular K+, and vice versa. We found DHO-blockade of Na/K pump current in canine ventricular myocytes also occurred with two affinities, which are very similar to those from guinea pig myocytes or rat ventricular myocytes. In contrast, isolated canine Purkinje myocytes have predominantly the type-h pumps, insofar as DHO-blockade and extracellular K+ activation are much closer to our type-h results than type-1. These observations suggest for mammalian ventricular myocytes: (a) the presence of two types of Na/K pumps may be a general property. (b) Normal physiological variations in extracellular pH and K+ are important determinants of Na/K pump current. (c) Normal physiological variations in the intracellular environment affect Na/K pump current primarily via the Na+ concentration. Lastly, Na/K pump current appears to be specifically tailored for a tissue by expression of a mix of functionally different types of pumps.

Gap junctional coupling in lenses from alpha(8) connexin knockout mice.

Lens fiber cell gap junctions contain alpha(3) (Cx46) and alpha(8) (Cx50) connexins. To examine the roles of the two different connexins in lens physiology, we have genetically engineered mice lacking either alpha(3) or alpha(8) connexin. Intracellular impedance studies of these lenses were used to measure junctional conductance and its sensitivity to intracellular pH. In Gong et al. 1998, we described results from alpha(3) connexin knockout lenses. Here, we present original data from alpha(8) connexin knockout lenses and a comparison with the previous results. The lens has two functionally distinct domains of fiber cell coupling. In wild-type mouse lenses, the outer shell of differentiating fibers (see 1, DF) has an average coupling conductance per area of cell-cell contact of approximately 1 S/cm(2), which falls to near zero when the cytoplasm is acidified. In the inner core of mature fibers (see 1, MF), the average coupling conductance is approximately 0.4 S/cm(2), and is insensitive to acidification of the cytoplasm. Both connexin isoforms appear to contribute about equally in the DF since the coupling conductance for either heterozygous knockout (+/-) was approximately 70% of normal and 30-40% of the normal for both -/- lenses. However, their contribution to the MF was different. About 50% of the normal coupling conductance was found in the MF of alpha(3) +/- lenses. In contrast, the coupling of MF in the alpha(8) +/- lenses was the same as normal. Moreover, no coupling was detected in the MF of alpha(3) -/- lenses. Together, these results suggest that alpha(3) connexin alone is responsible for coupling MF. The pH- sensitive gating of DF junctions was about the same in wild-type and alpha(3) connexin -/- lenses. However, in alpha(8) -/- lenses, the pure alpha(3) connexin junctions did not gate closed in the response to acidification. Since alpha(3) connexin contributes about half the coupling conductance in DF of wild-type lenses, and that conductance goes to zero when the cytoplasmic pH drops, it appears alpha(8) connexin regulates the gating of alpha(3) connexin. Both connexins are clearly important to lens physiology as lenses null for either connexin lose transparency. Gap junctions in the MF survive for the lifetime of the organism without protein turnover. It appears that alpha(3) connexin provides the long-term communication in MF. Gap junctions in DF may be physiologically regulated since they are capable of gating when the cytoplasm is acidified. It appears alpha(8) connexin is required for gating in DF.

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.

Spatial variations in membrane properties in the intact rat lens.

We have used linear frequency domain techniques to measure impedance at various locations and depths in the intact rat lens. The data are used to obtain best-fit solutions to a new electrical model based on lens structure, allowing us to estimate localized conductances of surface cell membranes (Gs), fiber cell membranes (gm), and gap junctions (Gj) as functions of position. We find that gm is small and fairly uniform throughout the lens (2.02 +/- 0.58 microS/cm2); for the anterior surface-epithelial cells Gs = 1.26 +/- 0.19 mS/cm2; for the posterior surface differentiating fiber cells Gs = 0.46 +/- 0.04 mS/cm2. Thus, Gs varies about the equator in a stepwise fashion. Gj between fiber cells at locations interior to 80% of the radius is fairly uniform (0.75 S/cm2); but in the outer 20% Gj varies smoothly and symmetrically from both poles (0.66 S/cm2) to equator (5.95 S/cm2). This pattern of variation in Gj is similar to the pattern of inward and outward currents reported by Robinson and Patterson (1983. Curr. Eye Res. 2:843-847). We therefore suggest that the nonuniform distribution of functional gap junctions, not the surface cell conductance or Na/K pumps, may be responsible for directing these current flows. Gap junctional uncoupling during exposure to elevated calcium and acidification was also examined. High calcium (20 mM, with the calcium ionophore A23187) produced modest (twofold) irreversible uncoupling along with large, irreversible decreases in membrane potential. We did not pursue this further. Acidification with 20 and 100% CO2-bubbled Tyrode's produced 5- and 15-fold reversible uncoupling, respectively, only in the outer 20% of the lens radius. The remaining inner 80% of the lens gap junctions seemed resistant to the acidification and did not uncouple.

Transient elevation of spontaneous release at the frog neuromuscular junction following acetylcholine iontophoresis.

Miniature end-plate currents (m.e.p.c.s) were recorded from frog neuromuscular junctions using a two-electrode voltage clamp. The m.e.p.c. frequency was temporarily elevated following 10s iontophoretic applications of acetylcholine (ACh) when the junctions were clamped at 100 mV. This post-ACh "burst" of quanta was also observed at unclamped junctions. At-100 mV, the intensity of the burst was proportional to the amount of current flowing across the end-plate during ACh iontophoresis, but usually did not reach its peak until the end-plate receptor channels were almost completely closed. Addition of 0.5 microM TTX to the Ringer's solution, or total replacement of sodium with guanidine, lithium, or methylamine did not inhibit the burst. No burst was observed in Ca2+-free, EGTA solutions, or in Ca2+-free solutions containing 2 mM Mn2+. Sr2+ effectively substituted for Ca2+. Addition of 2 mM Co2+ or Mn2+ to normal Ringer's did not inhibit the burst. Presynaptic muscarinic receptors did not obviously contribute to the burst, since it was not blocked by atropine, nor produced by oxotremorine or pilocarpine. The ACh analogs carbachol and acetyl-beta-methylcholine also produced the burst. The burst was highly dependent on the muscle membrane potential during the period of ACh iontophoresis, becoming more intense at potentials negative to -100 mV and disappearing at -60 mV. The critical importance of the post-synaptic membrane potential suggests that the burst may be due to an action of the muscle end-plate on the motor nerve terminal, possibly by the movement of an anionic substance through open end-plate receptor channels, but this hypothesis does not account for the delay of the burst until near the end of the ACh-induced end-plate current.

Physiological properties of the normal lens.

The lens is an avascular organ suspended between the aqueous and vitreous humors of the eye. The cellular structure is symmetric about an axis passing through its anterior and posterior poles but asymmetric about a plane passing through its equator. Because of its asymmetric structure, the lens has historically been assumed to perform transport between the aqueous and vitreous humors. Indeed, when anterior and posterior surfaces were isolated in an Ussing chamber, a translens current was measured. However, in the eye, the two surfaces are not isolated. The vibrating probe technique showed the current densities at the surface of a free-standing lens were surprisingly large, about an order of magnitude greater than measured in an Ussing chamber, and were not directed across the lens. Rather, they were inward in the region of either anterior or posterior pole and outward at the equator. This circulating current is the most dramatic physiological property of a normal lens. We believe it is essential to maintain clarity; hence, this review focuses on factors likely to drive and direct it. We review properties and spatial distribution of lens Na+/K+ pumps, ion channels, and gap junctions. Based on these data, we propose a model in which the difference in electromotive potential of surface versus interior cell membranes drives the current, whereas the distribution of gap junctions directs the current in the observed pattern. Although this model is clearly too simple, it appears to quantitatively predict observed currents. However, the model also predicts fluid will move in the same pattern as ionic current. We therefore speculate that the physiological role of the current is to create an internal circulatory system for the avascular lens.

Estimating the time course of evoked quantal release at the frog neuromuscular junction using end-plate current latencies.

The use of end-plate current (e.p.c.) latency measurements to estimate the time course of the stochastic probabilistic process governing evoked release was investigated in the sciatic nerve-sartorius muscle preparation of the frog, Rana pipiens. We also examined the possibility that the release of a quantum depresses or enhances the subsequent release of additional quanta. Muscle end-plates were voltage clamped at 3-4 degrees C. Quantal release was restricted to a short, or localized, region of the nerve terminal using Ca2+-free, EGTA Ringer solution and a Ca2+-filled micropipette. The number of e.p.c.s containing 0, 1, 2, etc. quanta were totalled and compared to numbers predicted using Poisson's theorem. The differences between the actual and predicted numbers of events were not significant at the nineteen junctions studied (P less than 0.05). The latency of the first quantum observed in several hundred e.p.c.s was measured and used to calculate an estimate, alpha 1(t), of the time-dependent, probabilistic process, alpha (t), governing all evoked quantal release (Barrett & Stevens, 1972b). In three experiments, all quantal latencies were measured to obtain the actual alpha (t). The alpha 1(t) function gave an excellent approximation of alpha (t) (P greater than 0.2), in real and simulated latency data. The latency of the second quantum in the e.p.c.s was measured and used to provide another estimate, alpha 2(t), of alpha (t). The alpha 2(t) function was lower (depressed) during the first few milliseconds of the evoked release period, relative to alpha 1(t). The difference was significant (P greater than 0.01) in all experiments. Our measurement procedures were tested using computer-generated 'e.p.c.s' containing randomly occurring 'quanta'. These tests showed that the early depression was due to inadequate detection of the second quantum in the e.p.c.s. The effect of Sr2+ on evoked release was examined using double-barrelled pipettes containing 1 M-SrCl2 and CaCl2 solutions. The major result was that the durations of alpha 1(t) and alpha 2(t) were equally lengthened in Sr2+, relative to Ca2+.

Facilitation and the conduction of the nerve action potential at the frog neuromuscular junction.

We have measured the time intervals (latencies) between the delivery of paired stimuli and the appearance of the extracellularly recorded nerve terminal action potentials (n.a.p.s) and end-plate currents (e.p.c.s) at different temperatures in the sciatic nerve, sartorius muscle preparation of the frog. When the interstimulus interval was 70 ms, the n.a.p. and e.p.c. latencies following the second stimulus of the pair were shorter than following the first at 10 degrees C and 15 degrees C, and facilitation of the e.p.c. was observed. The shortened latency was also observed at 20 degrees C by Barton and Cohen (1982), who suggested that this may be related to facilitation. At 5 degrees C, however, the n.a.p. and e.p.c. latencies following the second stimulus were longer than following the first, and facilitation was still observed. When a 30 ms interstimulus interval was used, the changes in latency were even greater at the temperatures studied. The changes in e.p.c. latency were due to changes in the conduction velocity of the n.a.p., not changes in synaptic delay. At all temperatures and interstimulus intervals studied, the larger amplitude n.a.p.s were conducted more slowly, however, facilitation was always observed. We conclude that changes in the amplitude and conduction velocity of the nerve action potential do not obviously affect facilitation at the frog neuromuscular junction.

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.

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.

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 beta-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 microM 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.

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.

Steady-state current flow through gap junctions. Effects on intracellular ion concentrations and fluid movement.

Double voltage clamp studies were performed on gap junctions contained in septal membranes of the earthworm median giant axon. The gap junctions exhibited no conductance changes in response to voltages imposed across either the septal membrane or the plasma membrane. However, the trans-septal current displayed a slow (10 s) relaxation in response to transjunctional voltage steps. The experimental evidence suggests that this relaxation is a polarization of the septum due to local accumulation/depletion of permeant ions. A theoretical analysis of this observation suggests that the applied electric field causes accumulation of impermeant anions on one side of the junction and depletion on the other, which leads to a change in concentration of permeant ions to maintain macroscopic electroneutrality. The change in concentration of permeant ions generates a transjunctional equilibrium potential that opposes junctional current flow. These results indicate that currents flowing through gap junctions can have an influence on the distribution of intracellular ions. Moreover, the theoretical analysis suggests that such currents will be accompanied by significant intracellular and intercellular water flow.

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.

The inhibitory effect of beta-stimulation on the Na/K pump current in guinea pig ventricular myocytes is mediated by a cAMP-dependent PKA pathway.

The beta-agonist isoproterenol (ISO) reduces the Na/K pump current (Ip) via beta-adrenergic receptors when the intracellular calcium concentration ([Ca2+]i) is below 150 nM [8]. In the present study, the intracellular signaling pathway was investigated with whole-cell patch-clamp of isolated guinea pig ventricular myocytes. The inhibitory effect of ISO could be mimicked by external application of the membrane-permeant cAMP analog chlorophenylthio-cAMP (0.5 mM), the phosphodiesterase inhibitor isobutyl-1-methylxanthine (IBMX, 100 microM), or the adenylyl cyclase activator forskolin (50 microM). Intracellular application of the synthetic peptide inhibitor of protein kinase A (PKA), PKI (5 microM), prevented the effect of ISO. These results suggest that the inhibitory effect of ISO on Ip is mediated via a phosphorylation step induced by a cAMP-dependent PKA pathway. Neither the non-specific protein kinase inhibitor H7 (100 microM) nor the protein phosphatase inhibitor calyculin A (0.5 microM) had any effect on Ip in the absence of ISO. However, H7 could increase Ip and calyculin A could reduce it in the presence of ISO (1 microM and 12 nM respectively). These results indicate that there is a low basal level of phosphorylation which makes the effects of H7 and calyculin A difficult to detect in the absence of an ISO-induced increase in phosphorylation level.

The role of MIP in lens fiber cell membrane transport.

MIP has been hypothesized to be a gap junction protein, a membrane ion channel, a membrane water channel and a facilitator of glycerol transport and metabolism. These possible roles have been indirectly suggested by the localization of MIP in lens gap junctional plaques and the properties of MIP when reconstituted into artificial membranes or exogenously expressed in oocytes. We have examined lens fiber cells to see if these functions are present and whether they are affected by a mutation of MIP found in CatFr mouse lens. Of these five hypothesized functions, only one, the role of water channel, appears to be true of fiber cells in situ. Based on the rate of volume change of vesicles placed in a hypertonic solution, fiber cell membrane lipids have a low water permeability (pH2O) on the order of 1 micron/sec whereas normal fiber cell membrane pH2O was 17 micron/sec frog, 32 micron/sec rabbit and 43 micron/sec mouse. CatFr mouse lens fiber cell pH2O was reduced by 13 micron/sec for heterozygous and 30 micron/sec for homozygous mutants when compared to wild type. Lastly, when expressed in oocytes, the pH2O conferred by MIP is not sensitive to Hg2+ whereas that of CHIP28 (AQP1) is blocked by Hg2+. The fiber cell membrane pH2O was also not sensitive to Hg2+ whereas lens epithelial cell pH2O (136 micron/sec in rabbit) was blocked by Hg2+. With regard to the other hypothesized roles, fiber cell membrane or lipid vesicles had a glycerol permeability on the order of 1 nm/sec, an order of magnitude less than that conferred by MIP when expressed in oocytes. Impedance studies were employed to determine gap junctional coupling and fiber cell membrane conductance in wild-type and heterozygous CatFr mouse lenses. There was no detectable difference in either coupling or conductance between the wild-type and the mutant lenses.

Gap junctional coupling in lenses lacking alpha3 connexin.

Fiber cells of the lens are interconnected by an extensive network of gap junctions containing alpha3 (Cx46) and alpha8 (Cx50) connexins. A specific role for these connexins in lens homeostasis is not known. To determine the contribution of these connexins to lens function, we used impedance techniques to study cell-to-cell coupling in lenses from homozygous alpha3 knockout (-/-), heterozygous (+/-), and wild-type (+/+) mice. Western blots and immunofluorescence data indicated that alpha8 remained at similar levels in the three classes of lenses, whereas alpha3 was approximately 50% of the normal level in the +/- lenses, and it was absent from the -/- lenses. Moreover, the data from +/+ lenses suggest that a cleavage of connexins occurs abruptly between the peripheral shell of differentiating fibers (DF) and the inner core of mature fibers (MF). The appearance of the cleaved connexins was correlated to a change in the coupling conductance. In -/- lenses the coupling conductance of MF was zero, and these fibers were depolarized by about 30 mV from normal (approximately -65 mV). The DF remained coupled, but the conductance was reduced to 30-35% of normal. However, the gap junctions in the DF of alpha3 -/- lenses remained sensitive to pH. We conclude that alpha3 connexin is necessary for the coupling of central fibers to peripheral cells, and that this coupling is essential for fiber cell homeostasis because uncoupled MF depolarize and subsequently become opaque.

alpha-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. alpha-Adrenergic receptors were activated with noradrenaline (NA) after blocking beta-adrenergic receptors with propranolol. Ip was measured as the current blocked by strophanthidin (Str). 2. Activation of alpha-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 microM [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 alpha-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 alpha1-adrenergic agonists, stimulate Ip in a similar manner to NA. Stimulation of Ip by NA was eliminated by prazosin, an alpha1-antagonist, but was unaffected by yohimbine, an alpha2-antagonist. 7. We conclude noradrenaline activates ventricular alpha1-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.

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

We have previously shown activation of alpha1-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 microM at 15 nM cytosolic [Ca2+] ([Ca2+]i) and 13 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 alpha1-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 alpha1-activation caused a maximum increase of 15% vs 30% via PMA, and alpha1-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 alpha1-adrenergic activation to Ip is entirely through PKC, however alpha1-activation may be coupled to a specific population of PKC whereas PMA is a more global agonist.

Isoform-specific regulation of the sodium pump by alpha- and beta-adrenergic agonists in the guinea-pig ventricle.

1. Guinea-pig ventricle was used in the RNase protection assays to determine which alpha-isoforms of the Na+-K+ pumps are present, and ventricular myocytes were used in whole cell patch clamp studies to investigate the actions of alpha- and beta-adrenergic agonists on Na+-K+ pump current. 2. RNase protection assays showed that two isoforms of the alpha-subunit of the Na+-K+-ATPase are present in guinea-pig ventricle. The mRNA for the alpha1-isoform comprises 82 % of the total pump message, the rest being the alpha2-isoform. 3. We have previously shown that beta-adrenergic agonists affect Na+-K+ pump current (Ip) through a protein kinase A (PKA)-dependent pathway. We now show that these beta-effects are targeted to the alpha1-isoform of the Na+-K+ pumps. 4. We have also previously shown that alpha-adrenergic agonists increase Ip through a protein kinase C (PKC)-dependent pathway. We now show that these alpha-isoform effects are targeted to the alpha2-isoform of the Na+-K+ pumps. 5. These results suggest the effects of adrenergic activation on Na+-K+ pump activity in the heart can be regionally specific, depending on which alpha-isoform of the Na+-K+ pump is expressed.