Comparison of metabolic and neuropathy profiles of rats with streptozotocin-induced overt and moderate insulinopenia.

To assess the relative roles of insulinopenia, hyperglycemia and dyslipidemia in pathogenesis of diabetic neuropathy, we compared plasma insulin, glucose and lipid metabolism and peripheral nerve function in rats with streptozotocin (STZ)-induced overt and moderate insulinopenia (hyperglycemic, STZ-HG; random glucose>11 mM and normoglycemic, STZ-NG rats). While being slightly insulinopenic, STZ-NG rats are metabolically not different from control, naive animals, by having normal glucose tolerance and normal levels of plasma glucose, glycated HbA1c, cholesterol and triglycerides. Two weeks following injection of STZ, STZ-HG but not STZ-NG rats had suppressed motor nerve conduction velocity, F-wave prevalence, withdrawal responses to heat and von Frey filament stimuli. In apparent correlation with plasma insulin level, both STZ-HG and -NG rats manifested exaggerated responses in paw pressure and colorectal distension tests. These data suggest that insulinopenia may play a leading role in the diabetic impairment of deep muscle and visceral afferent pathways while hyperglycemia/dyslipidemia may represent a key requirement for the onset and progression of electrophysiological nerve impairment and loss of superficial heat and tactile perception. STZ-NG rats offer a convenient model for the investigation of the short-term effects of insulinopenia on peripheral nerve function.

Stretch receptor-associated expression of alpha 3 isoform of the Na+, K+-ATPase in rat peripheral nervous system.

Expression of the neuronal alpha(3) isoform of the Na(+),K(+)-ATPase (alpha(3) Na(+),K(+)-ATPase) was studied in the rat peripheral nervous system using histological and immunohistochemical techniques. Non-uniform expression of the alpha(3) Na(+),K(+)-ATPase was observed in L5 ventral and dorsal roots, dorsal root ganglion, sciatic nerve and its branches into skeletal muscle. The alpha(3) Na(+),K(+)-ATPase was not detected in nerve fibers in skin, saphenous and sural nerves. In dorsal root ganglion 12+/-2% of neurons were immunopositive for alpha(3) Na(+),K(+)-ATPase and all these neurons were large primary afferents that were not labeled by Griffonia simplicifolia isolectin B4 (marker of small primary sensory neurons). In dorsal and ventral roots 27+/-3% and 40+/-3%, respectively, of myelinated axons displayed immunoreactivity for alpha(3) Na(+),K(+)-ATPase. In contrast to the dorsal roots, strong immunoreactivity in ventral roots was observed only in myelinated axons of small caliber, presumably gamma-efferents. In the mixed sciatic nerve alpha(3) Na(+),K(+)-ATPase was detected in 26+/-5% of myelinated axons (both small and large caliber). In extensor hallicus proprius and lumbricales hind limb muscles alpha(3) Na(+),K(+)-ATPase was detected in some intramuscular axons and axonal terminals on intrafusal muscle fibers in the spindle equatorial and polar regions (regions of afferent and efferent innervation of the muscle stretch receptor, respectively). No alpha(3) Na(+),K(+)-ATPase was found in association with innervation of extrafusal muscle fibers or in tendon-muscle fusion regions. These data demonstrate non-uniform expression of the alpha(3) isoform of the Na(+),K(+)-ATPase in rat peripheral nervous system and suggest that alpha(3) Na(+),K(+)-ATPase is specifically expressed in afferent and efferent axons innervating skeletal muscle stretch receptors.

Mechanical hyperalgesia in rats with chronic perfusion of lumbar dorsal root ganglion with hyperglycemic solution.

In diabetes, chronic systemic hyperglycemia is associated with pain and other symptoms of peripheral neuropathy. Evaluation of mechanisms causing these symptoms is complicated because of the overlap between the systemic effects of hyperglycemia and its toxic effects within the peripheral nervous system. To address this problem we developed a technique for chronic local in vivo perfusion of rat lumbar dorsal root ganglion (DRG) with a hyperglycemic solution. Osmotic pumps were filled with 30 mM glucose in physiological buffer and implanted in normal adult rats. The output of the catheter attached to the pump was positioned in a hole drilled through the right transverse process of the L(5) vertebrae to perfuse the corresponding DRG. Repetitive tests of foot withdrawal to mechanical stimuli have shown that chronic hyperglycemia localized to the L(5) DRG causes hyperalgesia in the hind limb innervated by perfused ganglion but not in the contralateral limb. Control experiments (DRG perfusion with 5 mM glucose or 5 mM glucose+25 mM mannitol solution) have shown that hyperglycemia-induced hyperalgesia can not be attributed to surgery-related injury or hyperosmolality of the ganglion-perfusing solution. These data demonstrate direct functional toxicity of hyperglycemia in the peripheral nervous system. This technique provides a new approach for in vivo study of chronic effects of physiologically active factors on DRG neuron function.

Functional Na+/K+ pump in rat dorsal root ganglia neurons.

Steady-state Na+/K+ pump current (Ip) in isolated adult rat dorsal root ganglia neurons was studied to determine if the plasma membrane Na+/K+ pump activity is uniform across the population of dorsal root ganglia neurons. Cells were voltage-clamped at -40 mV and holding current (Ih) was recorded using whole-cell patch-clamp techniques under conditions that stimulate the Na+/K+ pump (60 mM intracellular Na+ and 5.4 mM extracellular K+). Ip was defined as the 1 mM ouabain-sensitive fraction of Ih. Data suggest the existence of three subpopulations of dorsal root ganglia neurons having mean steady-state Ip densities of 1.6+/-0.1, 3.8+/-0.2 and 7.5+/-0.4 pA/pF. Neurons with small Ip had an average soma perimeter of 95+/-3 microm, while neurons with medium and large Ip density had significantly larger soma sizes (131+/-8 and 141+/-7 microm, respectively). Neurons with a large Ip density had a significantly lower specific membrane resistance (Rm; mean 4.0+/-0.3 kohm x cm2) than neurons with medium or small Ip density (19+/-6 and 31+/-6 kohm x cm2, respectively). Regardless of these differences, in all groups of neurons Ip had a low sensitivity to ouabain (Ip half inhibition by ouabain was observed at 80-110 microM). These data suggest that the Na+/K+ pump site density and/or its activity is not uniform throughout the dorsal root ganglia neuron population; however, this non-uniformity does not appear to relate to the functional expression of the different alpha isoforms of the Na+/K+ pump. The major functional Na+/K+ pump in the dorsal root ganglia neuron plasma membrane appeared to be the low ouabain affinity (alpha1) isoform.

Effects of linoleic acid metabolites on electrical activity in adult rat ventricular myocytes.

Leukotoxin (Lx), an epoxide derivative of linoleic acid, has been suggested to be a toxic mediator of multiple organ failure in burn patients and of acute respiratory distress syndrome. Lx production was recently shown during myocardial ischemia/reperfusion. However, a recent study suggested that to be toxic Lx must be metabolized to Lx-diol. In the present study, isolated adult rat ventricular myocytes were studied with the whole-cell patch-clamp technique to determine the effects of these compounds on cardiac electrical activity. Measurements of action potentials showed that neither linoleic acid nor Lx (100 microM) caused any significant changes in action potential properties. However, Lx-diol in the range of 10-100 microM produced a dose dependent increase in duration and a decrease in overshoot of the action potential. Subsequent voltage clamp experiments isolating Na current (INa) and transient outward K current (Ito) revealed that Lx-diol inhibited INa and Ito by about 80% at 100 microM, while linoleic acid and Lx had no effect on these currents at the same concentration. While Lx-diol produced the same inhibition of INa and Ito at 100 microM, its effects were more potent on Ito with significant inhibition at 10 microM. Lx-diol also hastened the activation kinetics of Ito but not INa. The action of Lx-diol was rapid (reaching steady state in 3-5 min) and was reversible in 5-10 min following washout. Thus, Lx-diol could favor arrhythmias or cardiac arrest in intact heart and may be responsible for the cardiac problems seen in systemic inflammatory response syndrome. These results further support the suggestion that Lx is not toxic in the heart but rather must be metabolized to Lx-diol to produce toxic effects on cardiac muscle.

Non-uniform expression of alpha subunit isoforms of the Na+/K+ pump in rat dorsal root ganglia neurons.

Tissue sections and antibodies selectively recognizing isoforms of the alpha subunit of the Na+/K+ pump were used to determine the expression of alpha1, alpha2 and alpha3 pump isoforms in the plasma membrane of adult rat dorsal root ganglia (DRG) neurons. There was no detectable membrane signal from DRG neurons that were probed with antibodies to the alpha2 isoform of the Na+/K+ pump. The alpha1 isoform of the Na+/K+ pump was found in most (77+/-4%) studied DRG neurons, regardless of cell size. Only 16+/-7% of the neurons expressed a detectable level of the alpha3 Na+/K+ pump and all were apparently from a subpopulation of large DRG neurons. Comparison of cell size distributions and a study of neurons identified in serial sections suggested that of the alpha3 positive DRG neurons about 75% coexpressed the alpha1 isoform of the Na+/K+ pump. These data show that the expression of the protein of the alpha subunit isoforms of the Na+/K+ pump is not uniform throughout the population of DRG neurons and that alpha1 is the predominant isoform in the plasma membrane of these neurons.

Effects of streptozotocin-induced diabetes on heart rate, blood pressure and cardiac autonomic nervous control.

Diabetes-associated alterations in resting heart rate and blood pressure have been demonstrated in clinical studies and in animal models of insulin-dependent diabetes mellitus (IDDM). These alterations may result from changes in the heart, vasculature or autonomic nervous system control. Using the streptozotocin- (STZ-) treated rat model of IDDM, the current study was designed to: (1) monitor changes in heart rate and blood pressure continually during a 10-week period in conscious unrestrained animals; and (2) determine if observed alterations in heart rate were mediated by changes in sympathetic and/or parasympathetic nervous control. Biotelemetry techniques were used. Heart rate and blood pressure were recorded 24 h a day at 10 min intervals before and after induction of diabetes. Diabetes was induced by i.v. administration of 50 mg/kg STZ. Resting autonomic nervous system tone was estimated by chronotropic responses to full-blocking doses of nadolol (5 mg/kg i.p.) and atropine (10 mg/kg i.p.). STZ-induced diabetes was associated with time-dependent reductions in heart rate and its circadian variation. Diastolic blood pressure and mean arterial pressure did not differ significantly when compared between control and STZ-treated animals; however, pulse pressure was diminished in diabetic rats. Chronotropic responses to both nadolol and atropine were blunted significantly in diabetic animals suggesting that resting levels of both vagal and sympathetic nervous tone to the heart were diminished. Heart rate in the presence of both nadolol and atropine was also decreased in diabetic rats. All effects observed following administration of STZ were reversed, at least in part, by insulin treatment. These results suggest that IDDM is associated with time-dependent reductions in resting heart rate and autonomic nervous control of cardiac function.

Calcium-inhibitable current in cultured embryonic chick cardiac myocytes: possibly via a novel chloride channel.

The role of extracellular Ca2+ (Ca(2+)o) in the modulation of cardiac Cl- currents (I(Cl)) such as those activated by cAMP or swelling is uncertain. The effects of Ca(2+)o and extracellular cadmium (Cd(2+)o) on Cl- currents in cultured chick cardiac myocytes were investigated in Na+- and K+-free internal and external solutions using the whole-cell patch-clamp technique. In the absence of Na+ and K+ internally and externally, the whole-cell current was predominantly I(Cl). In the absence of cAMP, removal of Ca(2+)o (+ 1 mM EGTA) resulted in an increase in the current that was suppressed by reduction of Cl(o)- with a rightward shift of the zero-current potential towards the CI- reversal potential. We designated this current as a Ca2+-inhibitable I(Cl). Addition of 0.5 mM Cd(2+)o with or without removal of Ca(2+)o also resulted in a 1.5- to 2.0-fold increase in I(Cl) that was attenuated by 1 mM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). Under similar conditions, I(Cl) activated by Cd(2+)o (in 1 mM Ca(2+)o solution) was not further increased by subsequent removal of Ca(2+)o, suggesting that addition of Cd(2+)o and removal of Ca(2+)o activated the same I(Cl). In contrast, exposure to 1 microM forskolin further enhanced I(Cl) in the presence of Cd(2+)o. With 10 microM cAMP in the pipette solution, Ca2+-inhibitable I(Cl) could be activated in myocytes that do not possess cAMP-activated Cl- channels, indicating that activation of Ca2+-inhibitable I(Cl) does not require cAMP. In the presence of cAMP, in cells that display the cAMP-activated I(Cl), removal of Ca(2+)o resulted in a further increase in I(Cl) comparable to the Ca2+-inhibitable I(Cl). The Ca2+-inhibitable I(Cl) was minimized when pipette solutions contained 1.5 microM Ca2+. These results suggest that removal of Ca(2+)o or application of Cd(2+)o activates a Ca2+-inhibitable I(Cl) that is distinct from the cAMP-activated I(Cl).

Adrenergic stimulation of Na/K pump current in adult rat cardiac myocytes in short-term culture.

Passive membrane properties, steady-state Na/K pump current (Ip) and modulation of Ip by adrenergic agonists were studied with patch-clamp techniques in adult rat ventricular myocytes that were freshly isolated or maintained in culture for 1-4 days. Freshly isolated (day 0) myocytes had a 1.7-1.8 times smaller specific membrane resistance compared with that of cells on any day in culture. From day 0 to 4 there was a progressive decrease in cell capacitance (-17.6 +/- 0.8 pF/day) without a parallel decline in cell dimensions. The pump current density (1.55 pA/pF) was stable over the 0-4 days in culture. In rod-shaped myocytes norepinephrine (NE) and isoproterenol (ISO) stimulated Ip in a dose-dependent manner, with an apparent affinity of 36 +/- 8 and 1.5 +/- 0.4 nM, and maximum stimulation of 0.65 +/- 0.02 and 0.57 +/- 0.02 pA/pF, respectively. Nadolol suppressed this effect, suggesting that it was mediated by beta-adrenergic receptor activation. An inverse relationship was found between steady-state Ip and the stimulation of Ip by NE. In contrast to what was shown in guinea pig cardiac myocytes, in rat myocytes isoproterenol stimulation of Ip was not increased by intracellular [Ca] and it did not change the Ip-membrane potential relation. These results show that in adult rat cardiac myocytes NE and ISO are potent stimulators of Na/K pump activity, and this effect may be studied using rat myocytes maintained in short-term culture.

Na(+)-K+ pump cycle during beta-adrenergic stimulation of adult rat cardiac myocytes.

1. The mechanisms underlying the increase in Na(+)-K+ pump current (Ip) caused by adrenergic stimulation were investigated in cultured adult rat cardiac myocytes using the whole-cell patch-clamp technique at 31-33 degrees C. 2. In myocytes perfused internally with 50 mM Na+ (0 K+i, 20 nM Ca2+, caesium aspartate solution) and externally with 5.4 mM K+o, noradrenaline (NA) and isoprenaline (Iso) (1-50 microM) stimulated Ip by 40-45%. 3. Na(+)-dependent transient Ip measurements with 0 mM K+i and 0 mM K+o revealed no change in the total charge transferred by the Na(+)-K+ pump during the conformational change, suggesting that the pump site density was not changed by adrenergic stimulation (2630 +/- 370 pumps micron-2 in control and 2540 +/- 190 pumps micron-2 in the presence of 10 microM NA). 4. With saturating Na+i or K+o (150 and 15-20 mM, respectively), Ip was still stimulated by NA and Iso. Thus, there was no indication that adrenergic activation of the Na(+)-K+ pump was mediated by accumulation of Na+i and K+o or changes in the Na(+)-K+ pump affinity for Na+i and K+o. 5. Both Ip and its increase under adrenergic stimulation were found to depend on [K+]i. While steady-state Ip decreased from 2.2 +/- 0.1 to 1.2 +/- 0.1 pA pF-1 (P < 0.05), the stimulation of Ip by 10 microM Iso increased from 0.38 +/- 0.04 to 0.67 +/- 0.06 pA pF-1 (P < 0.05) with an increase in [K+]i from 0 to 100 mM. 6. Under conditions that cause the Ip-Vm (membrane potential) relationship to express a positive slope ([Na+]o, 150 mM; [K+]o, 5.4 mM) or a negative slope ([Na+]o, 0; [K+]o, 0.3 mM) Iso stimulated Ip with no change in the shape of Ip-Vm curves. Thus, adrenergic stimulation of the Na(+)-K+ pump was not due to an alteration of voltage-dependent steps of the pump cycle. 7. Simulation of these data with a six-step model of the Na(+)-K+ pump cycle suggested that in rat ventricular myocytes a signal from adrenergic receptors increased the Na(+)-K+ pump rate by modulating the rate of K+ de-occlusion and release by the pump.

Diabetes with and without ketoacidosis on right atrial pacemaker rate and autonomic responsiveness.

Experiments were designed to determine whether insulin-dependent diabetes mellitus (IDDM) alters direct chronotropic effects of adrenergic and cholinergic agonists and whether the observed changes are associated with hyperglycemia or combined hyperglycemia and ketoacidosis. Diabetes was induced by intravenous administration of 45, 50, or 65 mg/kg streptozotocin (STZ). Rats treated with 65 mg/kg STZ had higher levels of blood glucose and ketones compared with the levels of the other groups. Right atria were isolated 12 wk after administration of STZ and bathed in Krebs-Henseleit solution. Basal spontaneous pacemaker rate was diminished in preparations isolated from diabetic rats. The maximum pacemaker rate observed during exposure to isoproterenol or norepinephrine was also depressed in preparations from diabetic animals; however, the increase in rate and half-maximal effective concentration values for each agent were not affected. The sensitivity to the negative chronotropic action of acetylcholine was enhanced by IDDM, whereas the response to carbachol (a cholinergic agonist not readily metabolized by acetylcholinesterase) was not changed. No significant differences were observed when we compared preparations isolated from diabetic animals with and without ketoacidosis. In summary, these data suggest 1) that IDDM is associated with a diminished basal spontaneous pacemaker without changes in the responsiveness to adrenergic and cholinergic receptor activation and 2) that ketoacidosis does not play a role in the observed alterations.

Na/K pump current in guinea pig cardiac myocytes and the effect of Na leak.

INTRODUCTION: Steady-state Na/K pump current (Ip) in adult guinea pig ventricular myocytes was studied to determine the effect on the Na/K pump of transmembrane Na leak, membrane potential, and pipette Na concentration. METHODS AND RESULTS: Using conventional whole cell, patch clamp techniques, Ip was identified as either Ko-sensitive or ouabain-sensitive current when most other membrane currents were inhibited. Control experiments showed that there were no Ko-sensitive currents other than Ip under the conditions of our experiments. Ip was found to be similar to that reported by others being voltage dependent between -130 and 0 mV and having a half maximal activation by Nai of 28 mM. Ouabain sensitivity was also measured, and it was found that there were two binding sites with the high affinity site comprising 5% to 10% of the total and having an apparent affinity 1000-fold higher than the low affinity site. Apparent affinity of both sites was shifted about 10-fold (higher affinity) by increasing Nai from 10 to 85 mM. When internally perfused with 0 Na solution, Na leak through the membrane was found to be linearly related to Na/K pump activity. In contrast to prior suggestions, Ip was not correlated with series resistance when there was a large transmembrane Na gradient. CONCLUSION: These data suggest that, under conditions of high transmembrane Na gradient, Na leak through the membrane plays a significant role in determining Na/K pump activity.

Characterization of the Na/K pump current in N20.1 oligodendrocytes.

Glial cell Na,K-ATPase is suggested to participate in extracellular K+ concentration ([K+]o) control by being activated when [K+]o rises in the brain. The extent of that activation directly depends on the Na/K pump affinity to [K+]o, intracellular Na+ ([Na+]i) and, indirectly on pump cycle regulation by membrane potential (Vm). In the present investigation, these Na/K pump properties were studied with the whole-cell patch-clamp technique in cultured mouse oligodendrocytes (N20.1 cell line). N20.1 cells possess ouabain-sensitive Na/K pump current (Ip) with a maximal density of 0.5-0.6 pA/pF (estimated for conditions of Na/K pump stimulation by saturating [Na+]i, [ATP]i, [K+]o and at positive Vm). This current was half-inhibited at 83 +/- 31 microM ouabain, and half-activated by [Na+]i of 9.6 +/- 1.1 mM, by [K+]o of 2.0 +/- 0.1 mM and by membrane potential at about -65 mV. High levels of nervous activity may increase [K+]o from 3 to 12 mM which would only increase Na/K pump current by 40% due to the direct effect of [K+]o. However, elevated [K+]o would also depolarize the glial cell membrane which would indirectly activate Ip and together with the direct effect of [K+]o would increase Ip as much as 2-2.5-fold. These data suggest that glial cell Na/K pump regulation by Vm may be an important factor in determining the participation of the Na/K pump in [K+]o homeostasis in the nervous system.

A novel Cl- conductance in cultured chick cardiac myocytes: role of intracellular Ca2+ and cAMP.

Cl- conductance in cultured embryonic chick cardiac myocytes was characterized using whole-cell patch clamp techniques. Following elimination of cation currents in Na(+)- and K(+)-free internal and external solutions, the basal whole-cell current was predominantly a Cl- current. Cl(-)-sensitive current (ICl) was defined as the difference between the whole-cell currents recorded in normal and low [Cl-]o when measured in the same cell. The whole-cell current in the absence or presence of 10 microM cAMP was time independent, displayed outward rectification with the pipette [Cl-] < 40 mM, and was not saturated with a physiological Cl- gradient. The Cl- current was also activated by 1 microM forskolin and inhibited by 0.3 mM anthracene-9-carboxylic acid (9-AC). Forskolin was less effective than cAMP (internal dialysis) in activating the Cl- current. The cAMP- or forskolin-activated and basal Cl- current were reasonably fit by the Goldman-Hodgkin-Katz equation. The calculated PCl in the presence of cAMP was increased by five- to sixfold over the basal level. In the presence of 5 mM EGTA to decrease free [Ca2+]i, the whole-cell current could not be stimulated by cAMP, forskolin or IBMX (0.1 mM). These data suggest that cultured chick cardiac myocytes have a low basal Cl- conductance, which, as in some mammalian cardiac ventricular myocytes, can be activated by cAMP. However, this study shows that the activation process requires physiological free [Ca2+]i.

Effect of Napip on K0 activation of the Na-K pump in adult rat cardiac myocytes.

To determine if environmental factors influence the external K (K0) dependence of Na-K pump current (Ip), we systematically varied internal (pipette) Na (Napip) and Na-K pump activity while measuring the K0 dependence in adult rat cardiac myocytes. For each Napip, reactivation of Ip by K0 was dose dependent. The maximal Ip (Ipmax) and apparent affinity for K0 binding to the Na-K pump (K0.5) increased as Napip increased. The results of making an equimolar substitution of tetramethylammonium for K and Cs, and partial Ip inhibition with ouabain, also showed that Ipmax and K0.5 increased as Napip increased. We simulated pump activity as a function of intracellular Na (Nai) and K0 using a cyclic model of the Na-K pump and found that the model predicts K0.5 for K0 binding increases as Na increases, even when the conditions are adjusted by removing pipette K and partial pump inhibition with ouabain.

Effects of cesium on spontaneous rate in right atria isolated from diabetic rats.

Previous studies have shown that streptozotocin (STZ) induced diabetes in rats is associated with a decrease in the spontaneous rate of isolated right atria. Present experiments were designed to determine whether this model of insulin-dependent diabetes mellitus alters the chronotropic actions of cesium (Cs+). Right atrial preparations were isolated from STZ-treated and diluent-treated control rats, and bathed in Krebs-Henseleit buffer at 37 degrees C; dose-dependent (0.2-10 mM) effects of Cs+ were examined by cumulative addition. Preparations isolated from diabetic rats had a slower beating rate before exposure to Cs+, and the negative chronotropic response to this cation was diminished in these tissues. In fact, at concentrations between 2 and 10 mM Cs+, spontaneous rate did not differ between the diabetic and control groups. These data suggest that the hyperpolarization-activated current, I(f), may play a role in the slower spontaneous pacemaker rate observed in right atria isolated from STZ-induced diabetic rats.

Effect of Nai on activity and voltage dependence of the Na/K pump in adult rat cardiac myocytes.

We have measured the voltage dependence of the Na/K pump in isolated adult rat cardiac myocytes using the whole-cell patch-clamp technique. In the presence of 1-2 mM Ba and 0.1 mM Cd and nominally Ca-free, Na/K pump current (Ip) was measured as the change in current due to 1 mM ouabain. Voltage dependence of Ip was measured between -140 and +40 or +60 mV using square voltage-pulse and voltage-ramp protocols, respectively. With 150 mM extracellular Na (Nao) and 5.4 mM extracellular K (Ko), we found that the Na/K pump shows a strong positive voltage dependence between -140 and 0 mV and is voltage independent at positive potentials. Removing Nao reduced the voltage dependence at negative potentials with no effect at positive potentials. When Ko was reduced, a negative slope appeared in the current-voltage (I-V) curve at positive potentials. We have investigated whether Nai (intracellular Na) might also affect the voltage dependence of Ip by varying Na in the patch pipette (Napip) between 20 and 85 mM. We found, as expected, that Ip increased markedly as Napip was raised, saturating at about 70 mM Napip under these conditions. In contrast, while Ip saturated near +20 mV and declined to about 40% of maximum at -120 mV, there was no effect of Napip under these conditions. In contrast, while Ip saturated near +20 mV and declined to about 40% of maximum at -120 mV, there was no effect of Napip on the voltage dependence of Ip. This suggests that neither Nai binding to the Na/K pump nor the conformational changes dependent on Nai binding are voltage dependent. These results are consistent with extracellular ion binding within the field of the membrane but do not rule out the possibility that other steps, such as Na translocation, are also voltage dependent.

Alpha 1b-adrenoceptor-mediated stimulation of Na-K pump current in adult rat ventricular myocytes.

The purpose of this study was to determine if myocardial alpha 1a-and/or alpha 1b-adrenoceptors are involved in the increase in Na-K pump current (Ip) elicited by alpha 1-adrenergic agonists. Single rat ventricular myocytes were isolated by enzymatic disaggregation. The whole cell patch-clamp technique was used to examine dose-dependent effects of phenylephrine (PE) on holding current (Ih) and to determine whether observed actions were mediated via alpha 1a-or alpha 1b-adrenergic receptors. To minimize the contribution of transsar-colemmal currents other than Ip to Ih, membrane voltage was held constant -40 mV, and cells were maintained in a Ca-free perfusate containing 1 mM Ba and 0.1 mM Cd. All experiments were conducted in the presence of 3 microM nadolol. PE elicited dose-dependent increases in Ih, with a peak effect of 0.57 +/- 0.03 pA/pF observed at 30 microM. The response to PE was dose dependently inhibited by prazosin and chloroethylclonidine and was totally eliminated by 1 mM ouabain. When used at doses selective for the alpha 1a-subtype, WB4101 failed to significantly antagonize the action of PE. These data suggest that the observed alpha 1-adrenoceptor-mediated increase in Ih in isolated rat ventricular myocytes is the result of an increase in Ip effected via stimulation of alpha 1b-adrenergic receptors.