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PubMed1986

Conductive properties of the rabbit outer medullary collecting duct: outer stripe.

Segments of the outer medullary collecting duct were dissected from the outer stripe of the rabbit kidney (OMCDo) and perfused in vitro. The conductive properties of the tubule epithelium and individual cell membranes were determined by means of cable analysis and intracellular voltage-recording microelectrodes. The transepithelial voltage (VT) and resistance (RT) averaged -10.7 +/- 2.5 mV, lumen negative, and 28.5 +/- 2.9 k omega X cm (n = 27), respectively. Two cell types could be defined by their electrophysiological properties. One cell type (n = 7) had a mean basolateral membrane voltage (Vbl) of -30.1 +/- 2.4 mV, a fractional resistance of the apical membrane (fRa = Ra/Ra + Rbl) near unity (0.99 +/- 0.01), and a predominantly Cl(-)-selective basolateral cell membrane. The second cell type (n = 27) had a mean Vbl of -63.7 +/- 2.7 mV, a fRa of 0.81 +/- 0.02, and a predominantly K+-selective basolateral cell membrane. The present study focused on defining the conductive properties of this latter cell type. Amiloride (10(-5) M) and BaCl2 (2 mM) were used as probes of apical cell membrane Na+ and K+ conductive pathways, respectively. Amiloride increased fRa from 0.80 +/- 0.02 to 0.98 +/- 0.01 (n = 12), whereas BaCl2 increased fRa from 0.77 +/- 0.03 to 0.82 +/- 0.03 (n = 9). The conductive properties of the basolateral cell membrane were assessed by ion substitutions of the bath solution. A 10-fold increase in the bath [K+] depolarized Vbl by 34.9 +/- 1.9 mV (n = 16) in less than 1 s, indicating that this membrane was predominantly K+ selective.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1986

Unidirectional potassium fluxes in renal distal tubule: effects of chloride and barium.

Low luminal concentrations of chloride stimulate net potassium secretion by the renal distal tubule, independent of changes in transepithelial voltage. These effects are not prevented by the luminal application of the potassium channel blocking agent barium. Because net potassium secretion comprises secretory and absorptive components, we sought to evaluate the effects of chloride and barium on unidirectional potassium fluxes in the renal distal tubule. In vivo microperfusion methods were used in anesthetized Sprague-Dawley rats. Perfusion solutions contained either 42K or 86Rb as tracers for potassium. Tracer efflux coefficients, indicating apparent potassium permeability, were similar when measured using either isotope. Net potassium flux was determined as the difference between perfusion and collected rate, and unidirectional absorptive potassium flux was calculated as the product of the mean luminal potassium concentration and the tracer efflux coefficient. During perfusion with a solution that resembled fluid normally arriving at the early distal tubule, the absorptive potassium flux was approximately 25% of the unidirectional secretory flux. Reducing lumen chloride concentration increased net potassium secretion, because blood-to-lumen potassium flux increased from 61 +/- 12.7 to 96 +/- 14.6 pmol/min. Barium reduced both absorptive and secretory fluxes but did not prevent the stimulation of net potassium secretion that occurs when luminal chloride concentration is reduced. Apparent potassium permeability during perfusion with a solution that resembled fluid normally arriving at the early distal tubule was 800 nm/s when corrected for voltage. Together with the results of previous experiments, these results are consistent with the presence of a secretory pathway linking potassium with chloride in the luminal membrane of cells of the distal tubule.

Animals↗
PubMed1989

K+ channel currents in basolateral membrane of distal convoluted tubule of rabbit kidney.

To examine the nature of ion-conductive pathways in the basolateral membrane of rabbit distal convoluted tubule (DCT), we recorded single-channel currents from the tubule segment isolated from collagenase-treated kidney. Using cell-attached patch pipettes filled with 130 mM KCl, 5.4 mM CaCl2, and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.4), we observed K+ channels in the basolateral membrane of DCT, having two different single-channel conductances of 48.7 +/- 1.4 (n = 9) and 60.6 +/- 1.4 pS (n = 7). Both types of channels were completely blocked by 0.1 mM BaCl2. Both channels have same open probability of approximately 0.5 at the intrinsic basolateral membrane voltage and were recorded with similar incidence. Mean open and closed times were 31.5 +/- 5.2 and 41.3 +/- 16.0 ms for the smaller channel, and 31.5 +/- 5.1 and 36.7 +/- 8.7 ms for the larger channel, respectively. These kinetic properties did not show any clear voltage dependence in both channels. Because of apparent similarity of channel kinetics, it is possible that both activities might represent different states of the same channel. For definite conclusion, however, further investigations are necessary. In three recordings from 54 successful patches, we observed a flickering channel with rapid kinetics, which was insensitive to 1 meq/l Ba2+. The conductance of this channel was 76.6 pS (n = 2). The extrapolated zero current voltage was 76.0 mV (n = 2), indicating that this channel is permeable to K+. From these results, we suggest that K+ channels constitute conductive pathways for K+ in the basolateral membrane of rabbit DCT.

Animals↗
PubMed1992

Effect of insulin on potassium secretion in rabbit cortical collecting ducts.

Insulin is known to play an important role in the regulation of extrarenal K homeostasis. Previous clearance studies have shown that insulin decreases urinary K excretion, but the responsible nephron segments have not been identified. In this microperfusion study, in vitro, the effect of insulin on K transport in the cortical collecting duct (CCD), which is thought to be an important segment for regulation of the final urinary K excretion, was investigated. Basolateral insulin (10(-6) M) significantly inhibited net K secretion by 20% (mean JK = -26.2 +/- 4.2 peq.mm-1.min-1 for controls compared with -21.1 +/- 3.4 with insulin, P less than 0.001) and depolarized the transepithelial voltage (VT, from -14.6 +/- 3.5 to -10.8 +/- 3.5 mV, P less than 0.005), recovery did not occur over 60 min. Insulin (10(-11)-10(-5) M) depressed K secretion and depolarized the VT in a concentration-dependent manner. The half-maximal concentration was 5 x 10(-10) M, which is within the physiological range of plasma insulin concentration. In tubules of deoxycorticosterone acetate-treated rabbits, insulin also produced a significant fall in K secretion (from -43.4 +/- 7.5 to -36.1 +/- 5.7 peq.mm-1.min-1, P less than 0.05). Although luminal Ba (2 mM) decreased K secretion (from -14.4 +/- 2.9 to -7.0 +/- 1.7 peq.mm-1.min-1), basolateral insulin (10(-6) M) inhibited K secretion further (to -4.7 +/- 1.3 peq.mm-1.min-1, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1993

Functional identification of H-K-ATPase in intercalated cells of cortical collecting tubule.

A K-dependent proton extrusion mechanism was investigated by means of fluorescence techniques in rabbit cortical collecting tubules. These experiments were performed in split opened tubules from normal animals exposed to the intracellular pH (pH(i)) indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. This preparation permitted the separate study of the intercalated cells (IC) from the principal cells (PC). In IC pH(i) recovery in response to an acute acid load was observed under Na-free conditions on addition of 5 mM K. This K-dependent recovery of pH(i) in the IC was only partial, but was Sch 28080 inhibitable (10(-5) M) and ouabain insensitive. This suggests the process is mediated by an H-K-adenosinetriphosphatase similar to that of gastric cells. The PC were capable of recovering from the acid load, but this Na-independent response was not blocked by the Sch 28080, suggesting some other mechanism for this result. In both cell types reintroduction of Na into the superfusate resulted in full recovery back to the initial pH(i), presumably the result of Na/H exchange.

Alkalies↗
PubMed1990

Circulatory responses to vasoconstrictor agents during passive heating in the rat.

The purpose of this study was to determine the actions of several pharmacological agents on the circulatory system, and more specifically on the superior mesenteric vascular bed, in response to environmental heat stress in chloralose-anesthetized rats. Animals were instrumented with Doppler flow probes on the mesenteric and renal arteries and exposed to an ambient temperature of 40 degrees C. Heart rate, mean arterial blood pressure (MAP), and core (Tc) and tail skin temperatures were also monitored. As Tc progressively increased from 37 degrees C during heat exposure, MAP rose to a plateau and then fell precipitously as Tc exceeded 41.5 degrees C. Mesenteric resistance increased throughout the early stages of heating before sharply declining prior to the reduction in MAP. The pressor and mesenteric resistance responses to constant infusions of several adrenergic agonists after MAP began falling (Tc = 41.3 degrees C) were significantly (P less than 0.05) attenuated compared with infusions into normothermic animals. In a second set of experiments, injections of both norepinephrine and angiotensin II were made 30 min before and approximately 10, 30, 50, 70, and 90 min after initiation of heating. These injections increased both MAP and mesenteric resistance; however, at TcS greater than 40 degrees C, the responses to both agonists were progressively and significantly attenuated. In a final group of animals, barium chloride infusions produced similar pressor and regional resistance changes during both normothermia and severe hyperthermia (Tc greater than 42 degrees C). These results indicate that, in the chloralose-anesthetized rat, hyperthermia disrupts adrenoceptor function but does not alter the intrinsic ability of vascular smooth muscle to contract.

Angiotensin II↗
PubMed1994

Release of creatine kinase and prostaglandin E2 from regenerating skeletal muscle fibers.

To study the effect of regeneration on the release of creatine kinase (CK) and prostaglandin E2 from muscle, extensor digitorum longus muscles of mice were injected with 50 microliters of BaCl2 solution in saline (1.2% wt/vol). Injected muscles showed almost complete degeneration at 3 days postinjection but had regenerated to approximately the same fiber cross-sectional area as contralateral control muscles by 12 days postinjection. These muscles released reduced amounts of intracellular CK compared with contralateral control muscles in response to excessive isometric contractile activity or treatment with the calcium ionophore A-23187 (20 microM) in vitro. However, regenerating muscles contained a lower total CK activity than contralateral control muscles, which accounted for the reduced CK efflux after experimental damage. Regenerating muscles released an increased amount of prostaglandin E2 compared with control muscles after both damaging stimuli. We conclude that regenerated muscles show no reduced susceptibility to contraction-induced damage (as assessed by CK release) but show an elevated release of prostaglandin E2 in response to contractile activity or an increase in intracellular calcium.

Animals↗
PubMed2002

Development of inward rectification and control of membrane excitability in mesencephalic v neurons.

The present study was performed to assess the postnatal development and functional roles of inward rectifying currents in rat mesencephalic trigeminal (Mes V) neurons, which are involved in the genesis and control of oral-motor activities. Whole cell voltage-clamp recordings obtained from Mes V neurons in brain stem slices identified fast (I(KIR)) and slow (I(h)) inward rectifying currents, which were specifically blocked by BaCl(2) (300-500 microM) or 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride (ZD 7288, 10 microM), respectively. The whole cell current density for these channels increased between postnatal days 2 to 12 (P2-P12), and the time courses for I(h) activation and deactivation were each well described by two time constants. Application of ZD 7288 produced membrane hyperpolarization in the majority of cells and prolonged afterhyperpolarization repolarization. Additionally, in the presence of ZD 7288, spike frequency was decreased and adaptation was more pronounced. Interestingly, these neurons exhibited a voltage-dependent membrane resonance (<10 Hz) that was prominent around resting potential and more negative to rest and was blocked by ZD 7288. These results suggest that I(h) contributes to stabilizing resting membrane potential and controlling cell excitability. The presence of I(h) imparts the neuron with the unique property of low-frequency membrane resonance; the ability to discriminate between synaptic inputs based on frequency content.

Action Potentials↗
PubMed1989

Intraretinal analysis of the threshold dark-adapted ERG of cat retina.

1. The dark-adapted electroretinogram (ERG) obtained with 4-s flashes near threshold and at low intensities has three clear negative components: 1) a fast negative response at stimulus onset, 2) a slow negative response that increases in amplitude for about 2 s, and 3) a negative-going OFF response. The fast negative response was previously shown to originate from the scotopic threshold response (STR) arising in the proximal portion of neural retina. We investigated the intraretinal origins of the other two negative components and found that they also have origins in neural retina proximal to the photoreceptors. 2. In this study the ERG evoked in response to diffuse illumination of the dark-adapted cat retina was recorded between a chlorided silver wire in the vitreous and a plate behind the eye. Extracellular field potentials were recorded simultaneously with a microelectrode placed intraretinally at different retinal depths. In some cases light-dependent changes in extracellular K+ concentration ([K+]o) were recorded with the K+-selective barrel of a double-barreled microelectrode. 3. The three negative-going ERG potentials increased in amplitude from threshold up to approximately 6.0 log q.deg-2.s-1. The potential during illumination then became nearly flat, and the negative OFF response was exaggerated. At higher intensities, positive-going PII (DC-component and b-wave) and the c-wave emerged, whereas the a-wave (negative-going) was usually below threshold. 4. The slow negative ERG response was generated in neural retina, rather than by the retinal pigment epithelium. The response was positive-going in the transretinal recordings obtained in subretinal space. It was not, however, related to two positive-going responses from distal retina previously described as contributing negative components to the ERG: the neural retina component of the c-wave, termed slow PIII, and the rod-receptor potential. The slow negative response had a slower time course and lower threshold than slow PIII, and, more importantly, it was present in the absence of the [K+]o decrease in subretinal space that causes slow PIII. Furthermore, APB (1.2 mM vitreal concentration) blocked the entire negative-going ERG, whereas at higher intensities it blocked PII but not the c-wave in the ERG or slow PIII in transretinal recordings. These results indicate that the negative-going ERG components near threshold and at low intensities all originated proximal to the photoreceptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Light-evoked increases in [K+]o in proximal portion of the dark-adapted cat retina.

1. The scotopic threshold response (STR) and slow negative response are negative-going potentials in the dark-adapted electroretinogram (ERG) of the cat eye that originate proximal to the photoreceptors and are present at threshold and with dim illuminations. The present paper examines the hypothesis that these events are associated with Müller cell responses to potassium released by proximal retinal neurons by measuring light-dependent changes in extracellular K+ concentration [( K+]o, in proximal retina. 2. Extracellular field potentials and changes in [K+]o, evoked in response to diffuse illumination of the dark-adapted retina, were recorded with a double-barreled K+-sensitive microelectrode placed in the retina at different depths. The vitreal ERG was recorded at the same time. 3. The dynamic range of the light-evoked increases in [K+]o, recorded in proximal retina from threshold to saturation, was strikingly similar to that of the STR and slow negative response. The threshold for the [K+]o increase was near that of the most sensitive ganglion cells, and the response saturated approximately 2.4 log units below rod saturation. Also, onset latencies and rates of rise for the increases in [K+]o and the field potentials in proximal retina followed similar functions of intensity. 4. The depth distribution of the light-evoked [K+]o increases resembled that of the STR. The increases in [K+]o were largest and fastest in proximal retina where the STR was largest. This was true both for very low intensity stimuli, below the threshold for PII (DC-component and b-wave) in distal retina, and for stimulus intensities near STR saturation, where PII (DC-component) was present. For stimuli of intensities near rod saturation, when the b-wave was present in distal retina, there was a small fast increase in [K+]o at light onset in distal retina and a slower increase in proximal retina. Both increases were truncated by spread of the large light-evoked decrease in [K+]o in subretinal space that is known to cause the c-wave in the ERG and slow PIII in neural retina. 5. The duration of the increases in [K+]o in proximal retina was more sustained than the proximal field potentials. In response to stimuli of 2-4 s, the field potential began to recover toward base line after about 300 ms, whereas the increases in [K+]o remained at maximal levels until stimulus offset.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Light-evoked changes in [K+]o in proximal portion of light-adapted cat retina.

1. The M-wave is a light-adapted response of proximal retina consisting of phasic negative field potentials at light onset and offset that are spatially tuned for small stimuli. We measured light-dependent changes in extracellular K+ concentration ([K+]o) in proximal retina to investigate the hypothesis that the M-wave originates from Müller cell responses to changes in [K+]o. 2. Extracellular field potentials, and changes in [K+]o evoked in response to circular spots of light flashed on steady backgrounds, were recorded with double-barreled K(+)-sensitive electrodes placed in the retina at different depths. 3. Increases in [K+]o during illumination and at light offset were maximal in proximal retina, with the On [K+]o increase located more proximally than the Off increase. The [K+]o increase during illumination consisted of a phasic and sustained response, whereas the Off [K+]o increase was predominantly phasic. The spatial tuning of the [K+]o increases was similar to the tuning of the field potentials. 4. The Off-field potential was larger than the On potential; it tended to be maximal more distally and was more sharply localized in retinal depth. Stimulus-response characteristics of the field potentials were not altered by intravitreal tetrodotoxin (TTX; 3.8 microM) sufficient to block retinal ganglion cell action potentials. 5. There were no rod contributions to the proximal [K+]o increases and field potentials recorded at the background illuminations used in this study (9.5-11.5 log q.deg-2.s-1). 6. An intravitreal injection of L- or DL-2-amino-4-phosphonobutyric acid (APB; 1 mM) was used to block On-system neuronal responses in proximal retina and isolate Off-system responses. After APB the [K+]o response consisted of a sustained decrease in [K+]o during illumination followed by an overshoot at light offset, while the field potential was a sustained positive response at light onset followed by an initially phasic negative response at light onset followed by an initially phasic negative response at light offset. These responses retained spatial tuning. To isolate the On-system components, the APB-isolated responses were subtracted from the controls. The [K+]o response now consisted of a sustained increase during illumination followed by an undershoot at light offset. The field potential was a sustained negative potential with an initial phasic peak that decayed at Off. Results with kynurenate (KYN; 5 mM) and (+/-)cis-2,3-piperidine (PDA; 5 mM) confirmed the sustained nature of the On component.(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptation, Physiological↗
PubMed1994

Variation in IH, IIR, and ILEAK between acutely isolated adult rat dorsal root ganglion neurons of different size.

1. The distribution of IH, IIR, and ILEAK was studied in different diameter rat dorsal root ganglion (DRG) neuron cell bodies (neurons). DRG neurons were studied in three diameter ranges: small (19-27 microns), medium (33-37 microns), and large (44-54 microns). IH was defined as a slowly activating inward current evoked by hyperpolarizing voltage steps from a holding potential (HP) of -60 mV, and blocked by 1 mM Cs2+ but not 1 mM Ba2+. Inward rectifier current (IIR) was defined as a rapidly activating current evoked by hyperpolarizations from HP -60 mV, which rectified inwardly around the reversal potential for potassium (EK), and was completely blocked by 100 microM Ba2+. ILEAK was defined as an outward resting current at HP -60 mV, which did not rectify and was blocked by 100 microM Ba2+ but not by 2 mM Cs+. 2. IH was observed in 23 of 23 large, 11 of 12 medium, and in 9 of 20 small diameter DRG neurons tested. Peak IH normalized to membrane surface area was significantly greater in large than in medium or small diameter DRG neurons expressing IH. All neurons exhibiting IH under voltage clamp conditions had short duration action potentials and exhibited time-dependent rectification under current clamp conditions, properties similar to A-type DRG neurons. The 11 small diameter neurons not expressing IH had long duration action potentials and did not exhibit time-dependent rectification, properties similar to C-type DRG neurons. 3. IIR was detected in 18 of 22 medium diameter neurons tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Hyperpolarization-activated inward current in neurons of the rat's dorsal nucleus of the lateral lemniscus in vitro.

Hyperpolarization-activated inward current in neurons of the rat's dorsal nucleus of the lateral lemniscus in vitro. J. Neurophysiol. 78: 2235-2245, 1997. The hyperpolarization-activated current (Ih) underlying inward rectification in neurons of the rat's dorsal nucleus of the lateral lemniscus (DNLL) was investigated using whole cell patch-clamp techniques. Patch recordings were made from DNLL neurons of young rats (21-30 days old) in 400 micro;m tissue slices. Under current clamp, injection of negative current produced a graded hyperpolarization of the cell membrane, often with a gradual sag in the membrane potential toward the resting value. The rate and magnitude of the sag depended on the amount of hyperpolarizing current. Larger current resulted in a larger and faster decay of the voltage. Under voltage clamp, hyperpolarizing voltage steps elicited a slowly activating inward current that was presumably responsible for the sag observed in the voltage response to a steady hyperpolarizing current recorded under current clamp. Activation of the inward current (Ih) was voltage and time dependent. The current just was seen at a membrane potential of -70 mV and was activated fully at -140 mV. The voltage value of half-maximal activation of Ih was -78.0 +/- 6.0 (SE) mV. The rate of Ih activation was best approximated by a single exponential function with a time constant that was voltage dependent, ranging from 276 +/- 27 ms at -100 mV to 186 +/- 11 ms at -140 mV. Reversal potential (Eh) of Ih current was more positive than the resting potential. Raising the extracellular potassium concentration shifted Eh to a more depolarized value, whereas lowering the extracellular sodium concentration shifted Eh in a more negative direction. Ih was sensitive to extracellular cesium but relatively insensitive to extracellular barium. The current amplitude near maximal-activation (about -140 mV) was reduced to 40% of control by 1 mM cesium but was reduced to only 71% of control by 2 mM barium. When the membrane potential was near the resting potential (about -60 mV), cesium had no effect on the membrane potential, current-evoked firing rate and input resistance but reduced the spontaneous firing. When the membrane potential was more negative than -70 mV, cesium hyperpolarized the cell, decreased current-evoked firing and increased the input resistance. Ih in DNLL neurons does not contribute to the normal resting potential but may enhance the extent of excitation, thereby making the DNLL a consistently powerful inhibitory source to upper levels of the auditory system.

Animals↗
PubMed1998

Whole cell patch-clamp recordings of rat midbrain dopaminergic neurons isolate a sulphonylurea- and ATP-sensitive component of potassium currents activated by hypoxia.

The effects of brief (2-4 min) hypoxia on presumed dopaminergic "principal" neurons of the rat ventral mesencephalon were investigated by using either intracellular or whole cell patch-clamp recordings in in vitro conditions. Under single-electrode voltage clamp, with sharp microelectrode (Vh -60 mV), a brief hypoxia caused an outward current (hypoOUT) of 110.2 +/- 15.2 (SE) pA (n = 18), which was followed by a posthypoxic outward current (posthypoOUT) of 149.6 +/- 10.6 pA (n = 18). Although the hypoOUT reversed at -83.7 +/- 3.8 mV (n = 18), the posthypoOUT did not reverse. The K+ATP-blocking sulphonylureas tolbutamide (100 microM) and glibenclamide (30 microM), significantly reduced the peak of the hypoOUT by 47.6 +/- 7.7% (n = 16) and 54.18 +/- 7.5% (n = 3), respectively. In contrast, they did not affect the posthypoOUT. Extracellular barium (300 microM to 1 mM) almost abolished the hypoOUT, leaving the posthypoOUT unchanged. The large K+ channel blocker charybdotoxin (10-50 nM), depressed the hypoOUT after tolbutamide treatment. To investigate whether or not cytosolic factors might control the development of the hypoOUT, we dialyzed the principal neurons by patch-clamp recordings (Vh -60 mV). Under whole cell recordings hypoxia evoked an hypoOUT of 70.2 +/- 14.5 pA that reversed polarity at -87.9 +/- 5.1 mV (n = 8). A small posthypoxic response was detected upon reoxygenation in a few neurons (4 out of 14). Three different sulphonylureas, tolbutamide (100 microM), glibenclamide (10-30 microM), and glipizide (100 nM) completely blocked the hypoOUT in patch-clamped neurons. The hypoOUT was also abolished by extracellular BaCl2 (300 microM). When the content of ATP in the dialyzate was raised from 2 to 10 mM no outward current/hyperpolarization was evoked by hypoxia. These data suggest that the hypoOUT, in principal neurons, is a complex response sustained by at least two barium-sensitive components: 1) an ATP-dependent, sulphonylurea-sensitive K+ conductance which could be isolated by the patch-clamp techniques and 2) a K+ conductance remaining after tolbutamide in intracellularly recorded neurons, which is sensitive to charybdotoxin and dependent on dialyzable cytosolic factors.

Adenosine Triphosphate↗
PubMed2000

Altered regulation of potassium and calcium channels by GABA(B) and adenosine receptors in hippocampal neurons from mice lacking Galpha(o).

To examine the role of G(o) in modulation of ion channels by neurotransmitter receptors, we characterized modulation of ionic currents in hippocampal CA3 neurons from mice lacking both isoforms of Galpha(o). In CA3 neurons from Galpha(o)(-/-) mice, 2-chloro-adenosine and the GABA(B)-receptor agonist baclofen activated inwardly rectifying K(+) currents and inhibited voltage-dependent Ca(2+) currents just as effectively as in Galpha(o)(+/+) littermates. However, the kinetics of transmitter action were dramatically altered in Galpha(o)(-/-) mice in that recovery on washout of agonist was much slower. For example, recovery from 2-chloro-adenosine inhibition of calcium current was more than fourfold slower in neurons from Galpha(o)(-/-) mice [time constant of 12.0 +/- 0.8 (SE) s] than in neurons from Galpha(o)(+/+) mice (time constant of 2.6 +/- 0.2 s). Recovery from baclofen effects was affected similarly. In neurons from control mice, effects of both baclofen and 2-chloro-adenosine on Ca(2+) currents and K(+) currents were abolished by brief exposure to external N-ethyl-maleimide (NEM). In neurons lacking Galpha(o), some inhibition of Ca(2+) currents by baclofen remained after NEM treatment, whereas baclofen activation of K(+) currents and both effects of 2-chloro-adenosine were abolished. These results show that modulation of Ca(2+) and K(+) currents by G protein-coupled receptors in hippocampal neurons does not have an absolute requirement for Galpha(o). However, modulation is changed in the absence of Galpha(o) in having much slower recovery kinetics. A likely possibility is that the very abundant Galpha(o) is normally used but, when absent, can readily be replaced by G proteins with different properties.

2-Chloroadenosine↗
PubMed1998

Effects of magnesium on human umbilical arteries: role of intracellular calcium stores.

The purpose of this investigation on human umbilical arteries was: (1) to analyze the effects of magnesium (Mg2+) in the prevention of contractions induced by 5-hydroxytryptamine (5-HT), KCl, Bay K 8644 and BaCl2; (2) to determine whether the presence of Mg2+ could modulate the response to caffeine in Ca2+-free medium at 25 and 37 degreesC, and (3) to examine the influence of Mg2+ and temperature in the release of intracellular calcium induced by 5-HT in Ca2+-free medium. Human umbilical arteries were dissected and mounted in organ baths for isometric measurement of force. Our results showed: (1) Mg2+ is a potent inhibitor of smooth muscle contraction of human umbilical arteries caused by 5-HT, KCl, Bay K 8644 and BaCl2, and that this effect might be due to the inhibition of the transmembrane calcium influx. (2) The relaxant effect of Mg2+ did not seem to be mediated by a blockade of the release of calcium from an intracellular store. (3) Moreover, our results suggest an effect of temperature independent from the action of Mg2+ on 5-HT-induced contraction.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1998

The peptide orphanin FQ inhibits beta-endorphin neurons and neurosecretory cells in the hypothalamic arcuate nucleus by activating an inwardly-rectifying K+ conductance.

Orphanin FQ (OFQ) is a novel heptadecapeptide whose structure resembles that of dynorphin A1-17. Its receptor shares appreciable homology with mu-, delta- and kappa-opioid receptors, and is highly expressed in the hypothalamus. The present study examined the effects of OFQ on neurons within the arcuate nucleus (ARC) of the mediobasal hypothalamus, using intracellular recordings from coronal slices. In current clamp, OFQ produced a hyperpolarization of ARC neurons, including those immunopositive for beta-endorphin, tyrosine hydroxylase and gonadotropin-releasing hormone. This hyperpolarization was dose-dependent, insensitive to antagonism by naloxone and was associated with a decrease in input resistance. In voltage clamp, OFQ produced an outward current associated with an increase in conductance. Varying the extracellular K+ concentration shifted the reversal potential for the OFQ response to the degree predicted by the Nernst equation. Furthermore, barium chloride markedly attenuated both the OFQ-induced hyperpolarization and decrease in input resistance. Administration of maximally effective concentrations of OFQ, followed by coadministration of maximal concentrations of either OFQ and the mu-opioid receptor agonist DAMGO or OFQ and the GABAB receptor agonist baclofen produced additive hyperpolarizations and outward currents. If DAMGO was applied first, followed by the coadministration of DAMGO and OFQ, then the responses were occluded. Taken together, these results indicate that OFQ inhibits beta-endorphin neurons, as well as A12 dopamine and GnRH neurosecretory cells, within the ARC by activating a subset of inwardly-rectifying K+ channels. This suggests that OFQ is not only an antiopioid peptide, but that it also modulates the hypothalamo-pituitary axis and, ultimately, reproductive behavior.

Animals↗
PubMed2001

GABA(B) receptors in anterior pituitary cells. Mechanism of action coupled to endocrine effects.

The activation of pituitary GABA(B) receptors by the specific agonist baclofen inhibits pituitary hormone secretion in vitro. Here we studied the mechanism of action of GABA(B) receptors in rat adenohypophysis. Anterior pituitary cells were obtained by trypsinization and were either plated for hormonal studies and cAMP determination or incubated in FURA 2AM for calcium measurements. Baclofen (BACL: 1 x 10(-5) M) significantly inhibited basal and thyrotropic releasing hormone (TRH)-stimulated (1 x 10(-7) M) PRL secretion in anterior pituitary cells from proestrous rats. In the presence of pertussis toxin (PTX: 150 ng/ml, 20 h), which leads to the uncoupling of the G(i/o)-protein from the receptor, both effects of BACL were abolished while the effect of dopamine (DA: 1 x 10(-8) M), used as an inhibitory control, was reduced from 70 to 25%. PTX also reversed BACL-induced inhibition of gonadotropin-releasing hormone (GnRH)-elicited luteinizing hormone (LH) secretion in anterior pituitary cells from 15-day-old female rats. In addition, though working in a pituitary mixed cell population, in which only some cell types possess GABA(B) receptors, BACL (1 x 10(-5) M) attenuated the forskolin-induced (0.5 microM) increase in cAMP. This effect was prevented by co-incubation with the antagonist 2 hydroxysaclofen and by preincubation with PTX. BACL (5 x 10(-5) M) and DA (5 x 10(-7) M) inhibited basal intracellular calcium concentrations ([Ca(2+)](i)) in pituitary cells and the effect of the latter was significantly stronger. The effect of BACL on [Ca(2+)](i) was abolished after preincubation with PTX. In the presence of the potassium channel blocking agents barium (200 microM and 1 mM) and tetraethylammonium (10 mM), BACL was still able to inhibit [Ca(2+)](i). Blockade of voltage-sensitive calcium channels (VSCC) with either verapamil (5 x 10(-6) M) or nifedipine (1 x 10(-6) M) completely abolished the effect of BACL on [Ca(2+)](i). In the presence of 12.5 mM potassium concentration baclofen significantly inhibited [Ca(2+)](i). In conclusion, our results describe the negative coupling of adenohypophyseal GABA(B) receptors to VSCC through PTX-sensitive G-proteins. These characteristics suggest a resemblance of these receptors to the typical presynaptic GABA(B) sites described in the central nervous system.

Animals↗