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The tetrodotoxin (TTX)-resistant Na(+) current arising from Na(V)1.8-containing channels participates in nociceptive pathways but is difficult to functionally express in traditional heterologous systems. Here, we show that injection of cDNA encoding mouse Na(V)1.8 into the nuclei of rat superior cervical ganglion (SCG) neurons results in TTX-resistant Na(+) currents with amplitudes equal to or exceeding the currents arising from natively expressing channels of mouse dorsal root ganglion (DRG) neurons. The activation and inactivation properties of the heterologously expressed Na(V)1.8 Na(+) channels were similar but not identical to native TTX-resistant channels. Most notably, the half-activation potential of the heterologously expressed Na(V)1.8 channels was shifted about 10 mV toward more depolarized potentials. Fusion of fluorescent proteins to the N- or C-termini of Na(V)1.8 did not substantially affect functional expression in SCG neurons. Unexpectedly, fluorescence was not concentrated at the plasma membrane but found throughout the interior of the neuron in a granular pattern. A similar expression pattern was observed in nodose ganglion neurons expressing the tagged channels. In contrast, expression of tagged Na(V)1.8 in HeLa cells revealed a fluorescence pattern consistent with sequestration in the endoplasmic reticulum, thus providing a basis for poor functional expression in clonal cell lines. Our results establish SCG neurons as a favorable surrogate for the expression and study of molecularly defined Na(V)1.8-containing channels. The data also indicate that unidentified factors may be required for the efficient functional expression of Na(V)1.8 with a biophysical phenotype identical to that found in sensory neurons.

The accurate determination of DNA concentration is essential for many processes in molecular biology and physiology and includes both gel- and cuvette-based methods. The recently introduced fluorescent dye, PicoGreen, has several advantages over other methods because it is sensitive and specific for double-stranded DNA (dsDNA). The dye is excited at 480 nm and emits at 520 nm when bound to dsDNA. This report describes the construction and use of PicoGmeter, a simple, inexpensive, fixed-wavelength fluorometer suitable for measuring PicoGreen fluorescence. PicoGmeter employs a blue light emitting diode (LED) for excitation and a photodiode to measure fluorescence. When compared to a commercially available instrument, PTI DeltaScan, the PicoGmeter performed admirably. Calibration curves for both instruments were superimposeable. Moreover, there was no significant difference between concentrations of DNA estimated by both instruments. A Bland and Altman analysis revealed that the PicoGmeter was equivalent to the PTI DeltaScan for estimating dsDNA concentration by the PicoGreen method. This simple, inexpensive, battery-operated fluorometer will allow investigators to employ the PicoGreen method without incurring the cost of purchasing a spectrofluorometer.

A variety of G-protein-coupled receptors regulate membrane excitability via M-type K(+) current (M-current) modulation. Muscarinic m1 and m3 acetylcholine receptors have both been implicated in the modulation of M-current. The muscarinic m5 receptor, like muscarinic m1 and m3 receptors, couples to phospholipase C via a pertussis toxin-insensitive G protein. Since a number of other receptors which activate phospholipase C also modulate M-current, we investigated if muscarinic m5 receptors could modulate recombinant M-type (KCNQ2/KCNQ3) K(+) channels after heterologous expression in human embryonic kidney (HEK) 293T cells. Application of Oxo-tremorine M to HEK293T cells expressing muscarinic m1, m3, or m5 receptors produced a similar robust inhibition of M-current, whereas muscarinic m2 and m4 receptor stimulation was without effect. Muscarinic m1, m3, or m5 receptor stimulation decreased the deactivation time constants of M-current at -50 mV. The inhibition of M-current by stimulation of muscarinic m1, m3, or m5 receptors was insensitive to overnight treatment with pertussis toxin or cholera toxin, which interfere with G(i/o) and G(s) G-protein signaling. These data suggest that muscarinic m1, m3, and m5 receptors inhibit M-channels via the activation of a common G protein.

The M-type potassium current (I(M)) plays a dominant role in regulating membrane excitability and is modulated by many neurotransmitters. However, except in the case of bradykinin, the signal transduction pathways involved in M-channel modulation have not been fully elucidated. The channels underlying I(M) are produced by the coassembly of KCNQ2 and KCNQ3 channel subunits and can be expressed in heterologous systems where they can be modulated by several neurotransmitter receptors including histamine H(1) receptors. In HEK293T cells, histamine acting via transiently expressed H(1)R produced a strong inhibition of recombinant M-channels but had no overt effects on the voltage dependence or voltage range of I(M) activation. In addition, the modulation of I(M) by histamine was not voltage sensitive, whereas channel gating, particularly deactivation, was accelerated by histamine. Non-hydrolysable guanine nucleotide analogues (GDP-beta-S and GTP-gamma-S) and pertussis toxin (PTX) treatment demonstrated the involvement of a PTX-insensitive G protein in the signal transduction pathway mediating histamine-induced I(M) modulation. Abrogation of the histamine-induced modulation of I(M) by expression of a C-terminal construct of phospholipase C (PLC-beta1-ct), which buffers activated Galpha(q/11) subunits, implicates this G protein alpha subunit in the modulatory pathway. On the other hand, abrogation of the histamine-induced modulation of I(M) by expression of two constructs which buffer free betagamma subunits, transducin (Galphat) and a C-terminal construct of a G protein receptor kinase (MAS-GRK2-ct), implicates betagamma dimers in the modulatory pathway. These findings demonstrate that histamine modulates recombinant M-channels in HEK293T cells via a PTX-insensitive G protein, probably Galpha(q/11), in a similar manner to a number of other G protein-coupled receptors. However, histamine-induced I(M) modulation in HEK293T cells is novel in that betagamma subunits in addition to Galpha(q/11) subunits appear to be involved in the modulation of KCNQ2/3 channel currents.

A variety of agonists are capable of inhibiting M-type K(+) channels via the activation of G-protein coupled receptors which converge on phospholipase-C (PLC) via the activation of G(q/11). Histamine acting via H(1) receptors also activates PLC but to date has not been shown to produce M-current (I(M)) modulation. We postulated that histamine would modulate recombinant M-channels after expressing histamine H(1) receptors in heterologous systems. Expression of KCNQ2 and KCNQ3 K(+) channel subunits by transient transfection in HEK 293T and HeLa cells caused the induction of a slow time-dependent outward current characteristic of I(M). Application of histamine (10 microM) to cells transfected with KCNQ2 and KCNQ3 K(+) channel subunits and H(1) histamine receptors produced a rapid and reversible inhibition. I(M) modulation by histamine was concentration-dependent, half maximal inhibition occurring at 399 nM with a Hill coefficient of 1.09 and was completely abolished by the H(1) receptor selective antagonist, astemizole. Studies of the modulatory effects of histamine on I(M) may aid in elucidating the signal transduction pathway(s) involved in I(M) modulation.

The present study was performed to determine whether renal efferent sympathetic neurons could be identified using a retrograde neuronal tracer without compromising renal function and whether the labeling and identification procedure alters Ca(2+) currents and neuromodulation of those neurons. Renal sympathetic and superior cervical ganglion (SCG) neurons were labeled with the fluorescent retrograde tracer fast blue. Renal function studies made 1 wk after labeling revealed that renal hemodynamics and fluid and electrolyte excretion were similar between the dye-injected (left) kidney and the control (right) kidney under control conditions and after hemorrhage. After volume expansion, urine flow in the dye-injected kidney was slightly, but significantly, less than that of the control kidney, whereas urinary sodium excretion increased by approximately ninefold in both kidneys. Patch-clamp studies of SCG neurons in 10 mM external Ca(2+) revealed that peak currents were not affected by the presence of the dye or a 1-min exposure to ultraviolet (UV) light. Neither maximal norepinephrine-induced Ca(2+) current inhibition nor the sensitivity to norepinephrine was affected by the dye or 1-min UV exposure. Facilitation protocols revealed that G protein modulation of Ca(2+) currents remained intact in dye-labeled UV-exposed neurons. This study demonstrates that a retrograde fluorescent dye technique to identify renal sympathetic neurons does not compromise renal function and the presence of the dye label or UV exposure has no effect on Ca(2+) currents and neuromodulation in these neurons. Isolation of single identified renal sympathetic neurons coupled with patch-clamp techniques represents a tool to investigate the role of individual current systems in the modulation of excitability in these neurons, which play an important role in the control of renal hemodynamics and excretory function and in the pathogenesis of hypertension.

Using whole-cell current-clamp measurements we have found that thapsigargin-mediated activation of store-regulated Ca2+ entry in rat basophilic leukemia cells is accompanied by complex changes in membrane potential. These changes consisted of: (i) an initial slow, small depolarization, (ii) a transitional change in potential to a depolarized value and (iii) transitional changes between a hyperpolarized and a depolarized potential. These complex changes in potential can be explained by the interaction between the endogenous inwardly rectifying K+ conductance and the generation of a small inward current. To investigate the possible influence of these changes of potential on [Ca2+]i, single cell measurements of fura2 fluorescence were undertaken alone or in combination with current-clamp measurements. Thapsigargin-mediated activation of the store-regulated Ca2+ entry pathway was accompanied by a marked increase of [Ca2+]i. During this increase, transient, abrupt declines in [Ca2+]i were detected in approximately 60% of the cells investigated. These changes of [Ca2+]i are consistent with the observed changes of membrane potential recorded under current-clamp.

The A-type K+ current (IA) of superior cervical ganglion neurons acutely isolated from spontaneously hypertensive (SHR) and age-matched Wistar-Kyoto (WKY) rats was compared under whole cell voltage clamp. Activation parameters were similar in each strain. Steady-state inactivation was shifted approximately -6 mV in SHR, where one-half inactivation occurred at -81 mV vs. -75 mV in WKY rats. The shift was not present in prehypertensive SHR but remained in adult enalapril-treated SHR and, therefore, may represent a primary alteration of IA properties. IA amplitudes evoked from physiological potentials were similar, despite inactivation of a greater fraction of the current in the SHR. Comparing maximal IA densities revealed that current density is elevated in the SHR, which compensates for the inactivation shift. Current density decreased with age in WKY neurons but did not significantly decline in SHR neurons unless hypertension was prevented with enalapril. Thus adult SHR neurons may retain a high IA density as an adaptive response to offset potential hyperexcitability resulting from the hyperpolarized IA inactivation.

We have characterized a Ca2+ current activated by depletion of intracellular Ca2+ stores (capacitative Ca2+ entry current) as a first step to investigate the mechanisms underlying communication between the intracellular Ca2+ stores and the plasma membrane Ca2+ permeability. Whole cell currents in response to voltage ramps from -125 to +60 mV from a holding potential of -40 mV were recorded in rat basophilic leukemia cells (RBL-1 cells) in solutions designed to optimize detection of a Ca2+ current. An inwardly rectifying current could be activated upon dialysis of the cell interior with pipette solutions devoid of Ca2+ and containing 20 mm BAPTA, a procedure expected to passively deplete intracellular Ca2+ stores. The current was maximally activated within 2 min, was sensitive to extracellular Ca2+ concentration and was abolished by removal of extracellular Ca2+. The current was markedly reduced in the presence of Ni2+ or La3+. The pathway activated by this protocol was permeant to Ba2+, displaying complex permeability characteristics at negative potentials. A small inward Mn2+ current consistent with a finite permeability of the pathway to Mn2+ was detected. In contrast Ni2+ displayed no detectable current carrying ability. Extracellular Na+ permeated the pathway in the absence of extracellular Ca2+. Under conditions designed to reduce passive depletion of intracellular Ca2+ stores, a Ca2+ current indistinguishable from that described above was activated by addition of ionomycin. This observation is consistent with the activation of the Ca2+ influx pathway occurring as a result of events associated with depletion of intracellular Ca2+ stores. Importantly, application of extracellular Ni2+ in the presence of ionomycin irreversibly inhibited the current. The presence of an inwardly rectifying K+ current in RBL cells could confound studies of the capacitative Ca2+ entry current when recorded using pipette solutions devoid of K+ since this current would be inward over the voltage range used to investigate the capacitative Ca2+ entry current. This study compares an inward rectifying K+ current and the capacitative Ca2+ entry current in RBL cells and highlights some similarities and differences between the two currents. The results demonstrate that caution should be exercised in interpreting recordings made using extracellular solutions containing even modest amounts of K+ when studying the capacitative Ca2+ entry current in RBL cells.

Whole-cell patch-clamp recordings were used to compare the amplitude and kinetics of acetylcholine-induced currents (IACh) in acutely isolated superior cervical ganglion (SCG) neurons from spontaneously hypertensive (SHR) rats and Wistar-Kyoto (WKY) rats, to determine if altered postsynaptic transmitter responsiveness underlies the increased sympathetic nerve activity in SHR rat neurons. Rapidly activating and slowly inactivating inward currents were recorded in response to rapid application of ACh (5 microM to 2 mM). Concentration/response relationships for SCG neurons isolated for SHR and WKY rats had dissociation constants of 161 microM and 169 microM, maximum responses of 26 nS/pF and 24 nS/pF, and Hill coefficients of 1.8 and 1.9, respectively. Activation of the currents was fitted well by a single exponential function with concentration-dependent time constants, whereas inactivation was fitted well by a double exponential function also with concentration-dependent time constants. The time constants of both activation and inactivation for SHR and WKY rats were not significantly different at any concentration tested. The results demonstrate that the postsynaptic effects of ACh are similar between SHR and WKY rat postganglionic neurons and, therefore, probably do not contribute to the observed differences in ganglionic transmission between SHR and WKY rat nerves.

1. The effects of NO donors on Ca2+ channel currents and noradrenaline (NA)-induced Ca2+ current inhibition were investigated in superior cervical ganglion (SCG) neurons using the whole-cell patch-clamp technique. 2. A 500 microM concentration of the NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP), enhanced Ca2+ current amplitude after either extracellular or intracellular application. The magnitude of Ca2+ current enhancement induced by NO donors was greater after intracellular application than after extracellular application. 3. Intracellular application of 1 mM guanosine 3',5'-cyclic monophosphate (cGMP) or 100 microM M&B 22948 (2-O-propoxyphenyl-8-azapurine-6-one), a cGMP phosphodiesterase inhibitor, or extracellular application of 1 mM 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) also increased the amplitude of Ca2+ currents thus mimicking the effect of the NO donors on Ca2+ channels. In contrast, pretreatment with Methylene Blue (100 microM) decreased the SNP (500 microM)-induced enhancement of Ca2+ currents. 4. Intracellular application of 500 microM SNP and SNAP, 100 microM M&B 22948 or 1 mM cGMP, or extracellular application of 200 microM 8-Br-cGMP reduced the magnitude of Ca2+ current inhibition induced by 5 microM NA. In addition, 500 microM SNP prevented the NA-induced shift of tail current activation curves to more depolarized potentials. 5. Internal dialysis with 500 microM SNP and SNAP or 1 mM cGMP, or extracellular application of 200 microM 8-Br-cGMP, reduced Ca2+ current facilitation produced by a depolarizing conditioning pulse both in the absence and presence of 5 microM NA. 6. The results suggest that NO donors induce enhancement of Ca2+ currents and block NA-induced Ca2+ current inhibition of SCG neurons via stimulation of cGMP formation.

An enhanced sympathetic nerve activity (SNA) has been implicated in the development and maintenance of the hypertension observed in experimental animal models such as the spontaneously hypertensive rat (SHR). Recent evidence suggests that an alteration of sympathetic synaptic transmission could also play a significant role in the elevation of SNA observed in the SHR (J. C. Magee and G. G. Schofield. Hypertension Dallas 20: 367-373, 1992). To test this hypothesis, we used intracellular recordings from superior cervical ganglion (SCG) neurons to compare properties of synaptic transmission between SHRs and two normotensive controls [Wistar-Kyoto (WKY) and Wistar rats]. Supramaximal preganglionic stimulation elicited larger amplitude fast excitatory postsynaptic potentials (EPSPs) and currents (EPSCs) in SHR sympathetic neurons compared with the normotensive controls. Analysis of variance of both compound and unitary EPSC amplitudes suggests that an increase in transmitter release is responsible for the elevated EPSP amplitude in these neurons. Also, a diminished short-term facilitation and an elevated synaptic depression limit the ability of SHR preganglionic neurons to increase transmitter release during short trains of repetitive stimuli. It is hypothesized that an enhanced transmitter depletion results in the altered synaptic plasticity of SHR sympathetic ganglion neurons. Intracellular recordings in low-Ca2+, high-Mg2+ external solutions support this idea. Therefore, synaptic transmission between the preganglionic and postganglionic neurons of SCGs from hypertensive rats was found to be altered in a manner that would tend to enhance sympathetic nervous activity, further implicating an exaggerated SNA in the pathogenesis of hypertension in the SHR model.

The effects of nitric oxide on the Ca2+ channel currents of adult rat superior cervical ganglion neurons were investigated using the whole-cell patch-clamp technique. Nitric oxide-containing compounds sodium nitroprusside and S-nitroso-N-acetyl-penicillamine increased the amplitude of Ca2+ currents by intracellular application. In addition, both sodium nitroprusside and S-nitroso-N-acetylpenicillamine reduced norepinephrine-induced inhibition of Ca2+ currents. The results suggest that nitric oxide might be involved in the modulation of Ca(2+)-dependent neurotransmission in sympathetic neurons.

1. Differences in the neuromodulation of Ca2+ currents between superior cervical ganglion (SCG) and more caudal paravertebral ganglion (PVG) neurons acutely isolated from the same rats were investigated using the whole-cell patch-clamp technique. 2. Norepinephrine (NE) induced a concentration-dependent inhibition of Ca2+ currents in both SCG and PVG neurons. The concentration producing 50% inhibition (IC50) for NE estimated from concentration-response curves was similar between SCG and PVG neurons but the maximal inhibition estimated from the concentration-response curve for PVG neurons was decreased compared with that of SCG neurons. 3. Tail current activation curves of both SCG and PVG neurons in the absence and presence of NE (5 microM) could be fitted to a double Boltzmann equation. In the presence of NE, the activation curves for both SCG and PVG neurons were shifted toward more depolarized potentials. The magnitude of the shift was greater in SCG than in PVG neurons, which could be accounted for by a greater decrease (P < 0.05) in the fractional amplitude of the first current component of SCG neurons (delta 1.4 +/- 0.4 nA, mean +/- SE, 39%) compared with that of PVG neurons (delta 0.9 +/- 0.1 nA, 16%). 4. Ca2+ current density, expressed as maximal tail current amplitude normalized to cell capacitance, was greater in PVG neurons than that in SCG neurons. 5. In SCG neurons, a saturating concentration of omega-conotoxin GVIA (omega-CgTx) produced a greater decrease of Ca2+ current amplitude at +20 mV (77.4 +/- 1.9%) than in PVG neurons (71.2 +/- 1.5%, P < 0.05). 6. After pretreatment with 15 microM omega-CgTx, NE still decreased the Ca2+ currents in both populations of neurons; however, the inhibition was greater in SCG neurons (31.1 +/- 3.4%) than in PVG neurons (12.8 +/- 3.6%, P < 0.01). 7. The dihydropyridine Ca2+ channel "agonist" Bay K 8644 (10 microM) prolonged Ca2+ tail currents in both SCG and PVG neurons. After normalizing to cell capacitance, there was no significant difference in Bay K 8644-induced tail current amplitude between the two populations of neurons. Moreover, NE (5 microM) increased the prolonged Ca2+ tail current amplitude induced by Bay K 8644 (10 microM) by 44.7 +/- 13.5% in SCG and 41.9 +/- 11.9% in PVG neurons. 8. Under control conditions, Ca2+ currents were facilitated by a depolarizing conditioning pulse (50 ms to +100 mV) in both PVG neurons (29.2 +/- 5.1%) and SCG neurons (20.1 +/- 4.0%).(ABSTRACT TRUNCATED AT 400 WORDS)

Experiments were performed to investigate if protein kinase C is involved in the norepinephrine-induced alpha 2-adrenoceptor-mediated inhibition of the Ca2+ current in adult rat superior cervical ganglion neurons. Ca2+ currents were recorded from dispersed superior cervical ganglion neurons, acutely isolated from adult rats, using the whole-cell patch-clamp technique. Both norepinephrine and the protein kinase C activator, 1,2-dioctanoylglycerol (diC8) decreased the Ca2+ current induced by step depolarizations to +10 mV from a holding potential of -80 mV. In the presence of norepinephrine, the Ca2+ current rising phase was adequately fit by a double exponential with a second time constant much larger than control, whereas in the presence of diC8 the rising phase became mono-exponential and the current displayed a prominent decay. Control tail current activation curves were described by the sum of two Boltzmann functions. Both norepinephrine and diC8 reduced peak tail current amplitude. Norepinephrine preferentially reduced the component activated at more hyperpolarized potentials, while diC8 preferentially reduced the component activated at more depolarized potentials. Intracellular application of three protein kinase C inhibitors: protein kinase C pseudosubstrate (PKC-19-36) (2 microM), staurosporine (1 microM) and 1-(5-isoquinolinylsulonyl)-2-sulfonylpiperazine (H-7) (50 microM), failed to affect norepinephrine-induced Ca2+ current inhibition. In addition, these protein kinase C inhibitors did not decrease the Ca2+ current inhibition induced by diC8.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate if Ca2+ currents of acutely isolated superior cervical ganglion (SCG) neurons are altered after dye labeling or ultraviolet (UV) exposure, SCG neurons were labeled by multiple injections of 2% fast blue (FB) (5-microliters total) into the submaxillary gland. Ca2+ currents of both labeled and unlabeled neurons were significantly depressed by 2-min exposure to UV, compared to labeled and unlabeled neurons not exposed to UV. The I-V curve of labeled neurons was shifted hyperpolarized after UV exposure. Tail-current activation curves of both labeled and unlabeled neurons were fitted to a double Boltzmann equation. In labeled neurons, 2-min exposure to UV produced a significant reduction of the amplitude of the more depolarized component as well as a hyperpolarizing shift of the half activation potential of both components of the activation curve. However, the short time (< 10 s) required to identify a target-specific neuron had no significant effect on the biophysical properties of Ca2+ currents of FB-labeled SCG neurons. Thus, use of this label in combination with the patch-clamp technique comprises a powerful approach to study membrane currents of target-specific neurons.

Transmission of neuronal activity was assessed by recording preganglionic and postganglionic compound action potentials in superior cervical ganglia isolated from adult spontaneously hypertensive rats (SHR), Wistar-Kyoto (WKY) rats, and Wistar rats as well as young SHR and WKY rats to determine if previously observed alterations of membrane excitability, synaptic transmission, or both, have an effect on the transmission of preganglionic activity in SHR. Single stimuli induced more postganglionic neurons to fire over a wide range of preganglionic stimulation intensities in superior cervical ganglia from adult SHR as compared with those from adult normotensive controls. Short stimulation trains confirmed that SHR are able to maintain this greater number of active postganglionic neurons during low-frequency stimulation (1-20 Hz). However, by the end of a train of high-frequency stimulation (70-100 Hz) fewer neurons fired in ganglia from SHR compared with those from normotensive controls. These differences in transmission were not observed in the young rats. The results from the present study demonstrate that physiological frequencies of preganglionic activity are more effectively transmitted through sympathetic ganglia from adult SHR compared with those from normotensive controls, and this enhanced transmission through ganglia may contribute to the elevated sympathetic activity and the consequent hypertension seen in this model.

The effects of norepinephrine on a Ca2+ current from acutely isolated and short-term (24 h) cultured adult rat superior cervical ganglion neurons were studied using the whole-cell variant of the patch-clamp technique. Norepinephrine produced a rapid, reversible and concentration-dependent reduction of the Ca2+ current. Accurately timed applications of norepinephrine (3 microM) showed that the development of Ca2+ current inhibition was delayed by up to 11 s after application of norepinephrine. Internal 500 microM guanylyl-imidodiphosphate (Gpp(NH)p) or guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S) decreased the Ca2+ current amplitude and induced a biphasic rising phase of the Ca2+ current. Under these conditions, the reduction of Ca2+ current amplitude by 3 microM norepinephrine was virtually abolished when compared with cells dialysed with GTP-containing internal solutions. Internal dialysis with solutions containing 2 mM guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S) increased the Ca2+ current amplitude and reduced the inhibition produced by 3 microM norepinephrine compared to cells dialysed with control internal solution. Treatment with 200 ng/ml pertussis toxin for 12-16 h greatly reduced the norepinephrine-induced Ca2+ current inhibition. Internal dialysis with solutions containing 500 microM cyclic adenosine 3',5'-monophosphate (cyclic AMP) and 3-isobutyl-1-methylxanthine had no significant effect on either the Ca2+ current inhibition by norepinephrine or the Ca2+ current amplitude. These results suggest that norepinephrine blocks a Ca2+ current in adult rat superior cervical ganglion neurons via a pertussis toxin-sensitive G-protein which is independent of intracellular cyclic AMP.

Isolated superior cervical ganglion (SCG) neurons were maintained in vitro at 22 degrees C (22 degrees C neurons) for up to 4 days in an effort to inhibit process outgrowth and thus extend the useful life of SCG neurons for voltage-clamp experiments. The neurons were viable after 4 days in vitro and remained roughly spherical whereas neurons maintained in vitro at 37 degrees C (37 degrees C neurons) developed extensive neurite processes after 2 days. The resting potential of 22 degrees C neurons was more hyperpolarized and the action potential duration was reduced compared to acutely isolated neurons (acute neurons) or 37 degrees C neurons. The steady state Na+ current activation and inactivation parameters of the acute and 22 degrees C neurons were similar whereas the half activation voltage was hyperpolarized and the slope factor of inactivation was reduced for the 37 degrees C neurons compared to the other two groups. The Na+ currents recorded from the 37 degrees C neurons displayed obvious signs of poor voltage control which were not observed in the acute or 22 degrees C neurons. The acetylcholine (ACh) sensitivity of both 22 degrees C and 37 degrees C neurons was significantly less than that of the acute neurons. This report demonstrates that room temperature culture of SCG neurons is a simple method which prevents process outgrowth and thus extends the useful life of the neurons for voltage-clamp experiments.

The effects of alpha-adrenoceptor agonist and antagonist drugs on the Ca2+ current of acutely isolated adult rat superior cervical ganglion (SCG) neurons were investigated to characterize the adrenoceptor which mediates a catecholamine-induced decrease of the Ca2+ current. Ca2+ currents were recorded using the whole-cell variant of the patch-clamp technique from neurons isolated enzymatically from adult rat SCG. Norepinephrine (1 microM) produced a rapid, reversible, and concentration-dependent decrease in Ca2+ current amplitude and slowed the rising phase of the Ca2+ current. These effects could be mimicked by clonidine (1 microM), an alpha 2-agonist but not by the alpha 1-agonist phenylephrine (1 microM). The norepinephrine-induced decrease in Ca2+ current amplitude was attenuated in the presence of idazoxan (1 microM), an alpha 2-antagonist, but was unaffected in the presence of the alpha 1-antagonist prazosin (1 microM). Neither antagonist displayed any Ca2+ current blocking activity. These results suggest that the alpha-receptor which mediates the norepinephrine-induced decrease of the Ca2+ current in adult rat SCG neurons is of the alpha 2-subtype.

No major differences were observed in the mechanical properties of diaphragm, extensor digitorum longus and soleus muscles from athymic nude and control mice. Denervated soleus muscles from nudes and controls showed no significant differences in their sensitivities to the cholinoceptor agonists acetylcholine and carbachol, either in the absence or presence of the anticholinesterase, physostigmine, suggesting that postjunctional receptor function is essentially normal. Phrenic nerve-diaphragm preparations from nudes were less sensitive to the twitch-augmenting effects of neostigmine. No difference in the time course of endplate potentials (epps) between nudes and controls was seen either in the absence or presence of neostigmine. Hence the observed differences in twitch augmentation are unlikely to be due to differences in acetylcholinesterase activity in the two muscles. In normal mice miniature endplate potential (mepp) amplitude decreased and mepp frequency increased with age. These changes were associated with an increase in muscle fibre diameter and a concomitant decrease in membrane resistance. Such changes did not occur in nude mice; thus mepp amplitude remained, high as in young normal muscle. It is suggested that the thymus may play a role in muscle development and that the effects on neuromuscular transmission are secondary to changes in development. In cut diaphragm muscles transmitter reversal potentials in nudes and controls were not different. Although there was no difference in the amplitude of the first epp of a train, or in the immediately releasable acetylcholine store, the quantal content of the first epp, the probability of transmitter release, the total nerve terminal acetylcholine store and the transmitter mobilization rate were all reduced. It is considered probable that all the measurable differences in transmitter release can be explained in terms of the nude muscle fibre diameter being small and being associated with a small nerve terminal size.

The aim of this study was to develop and characterize a model system in which the effects of ethanol on voltage- and agonist-gated ionic currents in adult mammalian neurons could be studied using voltage-clamp techniques. We have found that neurons enzymatically isolated from the peripheral (nodose and superior cervical ganglia) and central nervous system (pyramidal layer of the hippocampus) of the adult rat and guinea pig provide several advantages over conventional neuronal preparations (e.g., intact ganglia or brain slice). First, the isolated neurons, in conjunction with the patch clamp technique, allow high fidelity recordings of both macroscopic and single channel currents. Secondly, current- and voltage-clamp recordings have revealed that active and passive membrane properties, chemosensitivity, and ionic currents in the isolated neurons resemble those described from conventional preparations. Finally, we have developed an intracellular perfusion system which allows the convenient control of the intracellular milieu. This technique should be useful for the study of intracellular second messengers on ionic currents. Our results demonstrate that isolated adult mammalian neurons are ideally suited for the study of both the acute and chronic effects of ethanol on membrane excitability.

The actions of two clinically important dibenzocycloheptane antidepressant drugs, amitriptyline and nortriptyline, were studied on ionic channels of nicotinic acetylcholine (AcCho) receptors at the neuromuscular junction of frog skeletal muscle. Amitriptyline (5-10 microM) and nortriptyline (1-2 microM), like imipramine (5-10 microM), did not react with the nicotinic AcCho receptor but caused a voltage- and time-dependent decrease in the peak amplitude of the endplate current (epc). The time constant of epc decay, however, retained its voltage sensitivity. The voltage- and time-dependent effect of amitriptyline was nonlinear with regard to the current/voltage (I/V) relationship. Nortriptyline also had a more pronounced voltage- and time-dependent effect evidenced by a hysteresis loop in the I/V relationship of the epc was eliminated by the use of 50-msec stepwise changes of the membrane potential. The nonlinearity and hysteresis were due to a time-dependent phenomenon and did not involve previous AcCho receptor activation. The rate constant of the voltage- and time-dependent decrease in epc amplitude was sensitive to the membrane electric field and varied linearly with the membrane potential. Iontophoretically elicited epcs were much more depressed by both drugs than were spontaneous miniature epcs. There was no effect on the time constant of miniature epc decay, single-channel lifetime, or conductance. Thus (as we have pointed out in our histrionicotoxin studies) the primary site of action of these agents presumably is the activated but nonconducting species of the ionic channel of the nicotinic AcCho receptor. These agents, particularly nortriptyline, point to several different binding sites of the ionic channel and are suitable tools for the separation of the effects on peak current amplitude from its time constant of decay.

The electrical properties of nodose ganglion cells acutely isolated from adult rats were studied using the whole-cell patch-clamp recording method. Current-clamp recordings revealed a mean resting membrane potential of -54.3 mV and an input resistance of 527 M omega. Depolarizing current steps evoked action potentials with the following properties (mean): amplitude 111 mV, threshold -36 mV, and rate of rise 117 V/s. Two types of action potentials were observed, short and long duration. These properties, with the exception of input resistance (527 M omega cf. 50 M omega), are similar to those reported previously using intracellular recording methods in intact nodose ganglia (11, 20, 28). Brief application of 10 microM 5-hydroxytryptamine resulted in a rapid depolarization and burst of action potentials in the majority of cells. With voltage-clamp recording, step depolarizations to potentials positive to -10 mV elicited a transient inward current that was followed by a sustained outward current. Inward Na+ current was isolated by ion substitution and pharmacological agents. Two types of Na+ current were observed. One current was completely abolished by 3-15 microM tetrodotoxin (TTX), had a rapid time course, activated over the potential range -60 to -10 mV, and attained half-maximal conductance at -30 mV. The other current persisted in the presence of 15 microM TTX, had a slower time course, activated over the potential range -30 to 0 mV, and attained half-maximal conductance at -15 mV. In addition, 500 microM Cd2+ and 5.0 mM Co2+ reduced the TTX-insensitive current to 53 and 42% of control, respectively. Inward Ca2+ current was isolated by ion substitution and pharmacological agents and was identified by a dependence on external Ca2+. Cd2+ (500 microM) and Co2+ (5 mM) reduced the maximal inward current to 5 and 20% of control, respectively. When Ba2+ was substituted for Ca2+ as the charge carrier, the maximal inward current increased to 175% of control. Some cells had two Ca2+ current components, an inactivating component that activated near -60 mV and a large sustained current that activated near -40 mV. The initial inactivating current appeared as a "hump" on the current-voltage (I-V) curve over the potential range of -60 to -30 mV. The results indicate that, following isolation of these adult mammalian neurons, the membrane surfaces are sufficiently clean to allow patch-clamp recording.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of the muscarinic antagonist quinuclidinyl benzilate (QNB) on transmission at the frog sartorius neuromuscular junction have been examined. QNB decreases endplate potential (EPP) amplitude without affecting miniature endplate (MEPP) frequency or resting potential. QNB also increased the latency of the EPP and the nerve terminal spike in a frequency dependent fashion, suggesting the site of action is the unmyelinated nerve terminal. Since the rate and amplitude of muscle action are potentials decreased it is likely that QNB causes a blockade of electrically excitable sodium channels; the agent also blocks ionic channels associated with nicotinic acetylcholine receptors. It is possible that these effects of QNB may explain some of the behavioral disturbances produced by its administration.

Neurons enzymatically isolated from the adult rat superior cervical ganglion (SCG) were investigated using the whole-cell variant of the patch-clamp technique. Current-clamp studies revealed the following mean passive and active membrane properties: resting membrane potential, -54.9 mV; input resistance, 349 M omega; action potential (AP) threshold, -29.8 mV; AP overshoot, 53.3 mV; AP maximum rate of rise, 166.4 V/s; and AP duration, 3.2 ms. Chemosensitivity to acetylcholine remained intact following enzymatic dispersion. Voltage-clamp studies of a transient tetrodotoxin-sensitive Na+ current revealed activation and inactivation processes which could be fit to modified Boltzmann equations. Na+ current activation parameters for the half activation potential (Vh) and slope factor (K) were -23.3 mV and 5.3 mV, respectively. Inactivation parameters for Vh and K were -59.3 mV and 7.6 mV, respectively. Voltage-clamp studies also revealed a high voltage-activated sustained inward current which was eliminated upon removal of external Ca2+, greatly reduced by 500 microM Cd2+, and supported by Ba2+ or Sr2+. Tail current analysis of this Ca2+ current revealed a sigmoidal activation. A low voltage-activated transient Ca2+ current was not observed. We conclude that isolated SCG neurons retain the properties of neurons in intact ganglia and provide several advantages over conventional preparations for the study of voltage-gated membrane currents.

1. Monovalent cation selectivity and divalent cation sensitivity of the tetrodotoxin (TTX)-resistant Na+ current in dissociated adult rat nodose ganglion neurones were investigated using the whole-cell patch-clamp technique. 2. The TTX-resistant Na+ current was isolated using ion substitution and pharmacological agents. Under these conditions, the current reversal potential shifted 52 mV per tenfold change in external [Na+]. 3. Inorganic and organic monovalent cation permeability ratios (Px/PNa) were determined from changes in reversal potential and the Goldman-Hodgkin-Katz equation. The Px/PNa values determined by the former method were HONH3+, 1.38; Li+, 1.00; H2NNH3+, 0.66; NH4+, 0.28; CH3NH3+, less than 0.13; K+, less than 0.13; Rb+, less than 0.12; Cs+, less than 0.10; (CH3)4N+, less than 0.10. The values determined by either method agreed within 10%. 4. The effects of Cd2+, Co2+, Mn2+ and Ni2+ on the TTX-resistant Na+ current were analysed from peak-conductance values. These ions shifted the activation of the current to more positive potentials and decreased the maximal conductance. At 3 mM concentrations, Cd2+, Ni2+, Co2+ and Mn2+ decreased the maximal conductance 64.6, 50.7, 25.0 and 20.3%, respectively. 5. The results indicate that: (a) the monovalent cation selectivity of the TTX-resistant Na+ current is similar to that of the TTX-sensitive Na+ current in other tissues; and (b) the TTX-resistant Na+ current is less sensitive to divalent cations than the Ca2+ current in these neurones. These observations suggest that the structure determining the monovalent cation permeability of the TTX-resistant Na+ current is similar to that of the TTX-sensitive Na+ current in other tissues, and that the channels carrying the TTX-resistant Na+ current are distinct from those responsible for the Ca2+ current.

K+ currents of adult rat superior cervical ganglion neurons were studied using the voltage-clamp technique. Neuronal somata were dissociated from the ganglion using an enzymatic dispersion technique and voltage-clamped using the whole-cell patch-clamp technique. In solutions designed to isolate K+ currents, depolarization from a prepulse potential of -100 mV induced both transient and sustained outward current components. The transient current was completely eliminated by depolarization to -50 mV. The remaining sustained current component could be separated further into Ca2+-sensitive and Ca2+-insensitive components by superfusion with a Ca2+-free external solution. The transient current, which could be isolated by digital subtraction, rose rapidly and decayed over the subsequent 80 ms. Reversal potential determinations in different K+-containing solutions demonstrated that the current was carried primarily by K+. The transient current showed voltage-dependent inactivation, showing 50% inactivation near -87 mV and was completely inactivated at potentials more positive than -60 mV. The transient current recovered from inactivation with a voltage-dependent time course, the time course of inactivation decreasing with hyperpolarization. This transient outward current had characteristics of IA. The sustained Ca2+-insensitive outward current showed little decay over 800 ms and was also carried primarily by K+. This current component had characteristics similar to the delayed rectifier. A third sustained outward current eliminated by superfusion with Ca2+-free external solution had characteristics similar to the Ca2+-dependent K+ current.

The effects of the alkaloid pumiliotoxin-B were investigated on neurons from bullfrog paravertebral ganglia using current-clamp techniques. Pumiliotoxin-B (2 microM) induced repetitive action potential discharge or bursting pacemaker activity in response to a single stimulus. The toxin had no significant effect on the mean resting potential or action potential characteristics of single action potentials evoked prior to the action potential discharge onset. During the action potential discharge, action potential threshold and afterhyperpolarization amplitude were decreased. In the presence of pumiliotoxin-B, single action potentials were followed by a depolarizing afterpotential. Pumiliotoxin-B still induced action potential discharge in Ca2+-free or Cd2+-containing solutions. Brief superfusion with a Na+-free or tetrodotoxin-containing solution abolished the pumiliotoxin-B-induced action potential discharge prior to the blockade of directly elicited single action potentials. These solutions decreased or abolished the depolarizing afterpotential. Pumiliotoxin-B increases membrane excitability and can induce a stimulation-dependent action potential discharge which appears to result from a tetrodotoxin-sensitive Na+-sensitive potential.

The effects of neuropeptide Y (NPY) on the Ca2+ current of enzymatically dispersed neuronal somata from bullfrog paravertebral sympathetic ganglia were investigated using the patch-clamp technique. In C neurons, NPY induced a concentration-dependent decrease in the amplitude of the Ca2+ current and induced a slow biphasic current rising phase. Upon removal of NPY these parameters returned to near control values. NPY had no discernable effect on the Ca2+ current recorded in B neurons.

Preparations from alloxan diabetic rats showed a reduced sensitivity to the neuromuscular blocking action of (+)-tubocurarine but no alteration in sensitivity to the depolarizing neuromuscular blocking drug decamethonium. Physostigmine was less effective in augmenting twitch height in preparations from alloxan diabetic rats and such preparations had a significantly lowered total cholinesterase activity compared with control preparations. An additional observation was a reduction in the effectiveness of the pre-junctionally active agent beta-bungarotoxin in producing neuromuscular blockade in physostigmine-treated preparations from alloxan diabetic rats. All the changes produced by alloxan administration were prevented by treatment with insulin.

Single acetylcholine (ACh) channel currents were studied by the gigaohm patch-clamp technique in cultured sympathetic neurons of the bullfrog, Rana catesbeiana. Recordings were made at 22 degrees C on cell-attached and excised membrane patches. When ACh (0.5-1 microM) was present in the pipette, a single class of inward currents was observed with a chord conductance of 30 pS and a reversal potential of -2 mV. The mean channel open time was 11.6 ms at -65 mV and showed little or no voltage-dependence over the range -85 to -45 mV. These channels appear to mediate the fast nicotinic excitatory postsynaptic current.

To characterize further the somastatin (SOM) receptor mediating Ca2+ current reduction in rat superior cervical ganglion (SCG) neurons, the effects of three synthetic SOM octapeptide analogs, D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 (IM-4-82), D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 (DC 13-116), and D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-OL (SMS 201-995), which bind preferentially to pituitary SOM receptors (SOMa) were investigated. Ca2+ currents were recorded using the whole-cell variant of the patch-clamp technique from neurons isolated enzymatically from adult rat SCG. Application of the SOM analogs (0.003-3 microM) produced a rapid, reversible, and concentration-dependent decrease in Ca2+ current amplitude in addition to slowing the rising phase of the Ca2+ current. Estimates of the concentration producing half-maximal block (EC50) and maximum attainable block (Bmax) for DC 13-116, IM 4-28, and SMS 201-995 were 196, 67, and 9.5 nM, respectively, and 52, 57, and 48%, respectively. The results suggest that the SOM receptor on SCG neurons more closely resembles the SOMa receptor of the anterior pituitary than the SOMb receptor of cerebral cortical membranes.

1. The effects of somatostatin and somatostatin analogues on a Ca2+ current from acutely isolated and short-term (24-48 h) cultured adult rat superior cervical ganglion (SCG) neurones were studied using the whole-cell variant of the patch-clamp technique. 2. [D-Trp8]Somatostatin (SOM) produced a rapid, reversible and concentration-dependent reduction of the Ca2+ current. Ca2+ current amplitude was reduced over the voltage range -15 to +40 mV with the greatest reduction occurring where the amplitude was maximal (ca +10 mV). In the presence of SOM, the Ca2+ current rising phase was slower and biphasic at potentials between 0 and +40 mV. 3. Application of 0.1 microM-SOM for greater than 10 s resulted in a desensitization of the response. During a 4 min application of 0.1 microM-SOM, Ca2+ current amplitude returned to about 90% of control. A second application of 0.1 microM-SOM produced less block than the initial application. 4. Concentration-response curves for SOM, somatostatin-14 (SOM-14) and somatostatin-28 (SOM-28) were fitted to a single-site binding isotherm. The concentrations producing half-maximal block and the maximal attainable blocks of the Ca2+ current for SOM, SOM-14 and SOM-28 were 3.3, 5.4 and 35 nM, respectively and 55, 51 and 54%, respectively. SOM-14 and SOM-28 slowed the Ca2+ current rising phase in a manner similar to that of SOM. Somatostatin-28 had no effect on the Ca2+ current at 1 microM. 5. The magnitude of the Ca2+ current block produced by 0.1 microM-SOM was not significantly altered in the presence of 1 microM-idazoxan, atropine, naloxone or the somatostatin antagonist aminoheptanoyl-Phe-D-Trp-Lys-O-benzyl-Thr. 6. Internal dialysis with solutions containing 500 microM-guanylyl-imidodiphosphate (Gpp(NH)p) or guanosine-5'-O-(3-thiotriphosphate)(GTP-gamma-S) decreased the Ca2+ current amplitude by 36 and 41%, respectively, and induced a biphasic rising phase in the Ca2+ current. Under these conditions, application of 0.1 microM-SOM produced significantly less block of Ca2+ current amplitude (7.1 and 14.7%, respectively) when compared with controls. 7. Internal dialysis with solutions containing 500 microM-guanosine-5'-O-(2-thiodiphosphate)(GDP-beta-S) had no significant effect on either the Ca2+ current amplitude or block produced by 0.1 microM-SOM. 8. Internal dialysis with solutions containing 500 microM-cyclic adenosine 3',5'-monophosphate (cyclic AMP) and 3-isobutyl-1-methylxanthine had no significant effect on either the Ca2+ current block produced by 0.1 microM-SOM or the Ca2+ current amplitude.(ABSTRACT TRUNCATED AT 400 WORDS)

Somatostatin-like immunoreactivity has been reported to occur in the postganglionic neurons of sympathetic ganglia. We therefore investigated the effect of somatostatin (SOM) on the Ca2+ current in sympathetic neurons. Voltage-clamp recordings, using the whole-cell patch-clamp technique, were made from acutely isolated adult rat superior cervical ganglion (SCG) neurons in solutions (external and internal) designed to isolate Ca2+ currents. Application of 0.001-1.0 microM [D-Trp8]SOM resulted in a rapid, reversible and concentration-dependent decrease in the amplitude of the Ca2+ current evoked from a holding potential of -80 mV. The concentration-response relationship for SOM could be fitted to a single-site binding model with an apparent dissociation constant of 11 nM; the maximal attainable block of Ca2+ current by SOM was 50%. SOM also produced a pronounced slowing of the Ca2+ current rising phase, especially at more depolarized potentials. At higher concentrations (0.03-1.0 microM), prolonged application of SOM resulted in a progressive decrease in blocking ability. The results are consistent with a neurotransmitter and/or neuromodulator role for SOM in the sympathetic nervous system.