Integration of NPY, AGRP, and melanocortin signals in the hypothalamic paraventricular nucleus: evidence of a cellular basis for the adipostat.

Energy stores are held relatively constant in many mammals. The circuitry necessary for maintaining energy homeostasis should (1) sense the amount of energy stored in adipose tissue, (2) sense and integrate the multiple opposing signals regarding nutritional state, and (3) provide output regulating energy intake and expenditure to maintain energy homeostasis. We demonstrate that individual neurons within the paraventricular nucleus of the hypothalamus (PVH) are capable of detection and integration of orexigenic (neuropeptide Y [NPY]) and anorexigenic (melanocortin) signals, that NPY and melanocortins are functional antagonists of each other within the PVH in the regulation of feeding behavior, and that melanocortin administration within the PVH regulates both feeding behavior and energy expenditure. These data provide a cellular basis for the adipostat within neurons in the PVH that appear to be jointly regulated by NPY- and melanocortin-responsive neurons.

Measurements of intracellular calcium in sensory neurons of adult and old rats.

Cytosolic free calcium ([Ca2+]i) was measured using fluorescent digital imaging microscopy in rat dorsal root ganglion neurons isolated from animals of two age groups (adult: seven months; and old: 30 months). Neurons were enzymatically isolated and maintained in primary culture for 14 days. Cultured neurons were loaded with the fluorescent dye, Fura-2. The spatial distribution of resting [Ca2+]i was even in both adult and old rats, but the value of cytoplasmic free calcium in old neurons was significantly higher (207 +/- 37 nmol/l vs 96 +/- 23 nmol/l) in comparison with adult ones. Depolarization with 50 mmol/l K+ produced a rapid increase in [Ca2+]i in all neurons, but the values of depolarization-induced increase of [Ca2+]i in old neurons were significantly lower (423 +/- 54 nmol/l) compared with cells isolated from adult rats (1011 +/- 91 nmol/l). The time of the complete restoration of [Ca2+]i to the resting level was 10-times longer in old neurons. The caffeine-induced rise of intracellular calcium was somewhat higher in neurons from old animals, and its restoration to normal level was delayed. The findings indicate a substantial alteration of the mechanisms of regulation of intracellular calcium homeostasis with neuronal ageing.

Caffeine-induced calcium release from internal stores in cultured rat sensory neurons.

Free intracellular calcium concentration ([Ca2+]in) was recorded at 22 degrees C by means of Indo-1 or Fura-2 single-cell microfluorometry in cultured dorsal root ganglion neurons obtained from neonatal rats. The resting [Ca2+]in in dorsal root ganglion neurons was 73 +/- 21 nM (mean +/- S.D., n = 94). Fast application of 20 mM caffeine evoked [Ca2+]in transient which reached a peak of 269 +/- 64 nM within 5.9 +/- 1.1 s. After reaching the peak the [Ca2+]in level started to decline in the presence of caffeine and for 87.2 +/- 10.6 s cytoplasmic calcium returned to an initial resting value. In 40% of neurons tested [Ca2+]in decreased to subresting levels following the washout of caffeine (the so-called post-caffeine undershoot). On average, the undershoot level was 19 +/- 2.5 nM below the resting [Ca2+]in value. Prolonged exposure of caffeine depleted the caffeine-sensitive stores of releasable Ca2+; the degree of this depletion depended on caffeine concentration. The depletion of the caffeine-sensitive internal stores to some extent was linked to calcium extrusion via La(3+)-sensitive plasmalemmal Ca(2+)-ATPases. The stores could be partially refilled by the uptake of cytoplasmic Ca2+, but the complete recovery of releasable Ca2+ content of the caffeine-sensitive pools required the additional calcium entry via voltage-operated calcium channels. Caffeine-evoked [Ca2+]in transients were effectively blocked by 10 microM ryanodine, 5 mM procaine, 10 microM dantrolene or 0.5 mM Ba2+, thus sharing the basic properties of the Ca(2+)-induced-Ca2+ release from endoplasmic reticulum. Pharmacological manipulation with caffeine-sensitive stores interfered with the depolarization-induced [Ca2+]in transients. In the presence of low caffeine concentration (0.5-1 mM) in the extracellular solution the rate of rise of the depolarization-triggered [Ca2+]in transients significantly increased (by a factor 2.15 +/- 0.29) suggesting the occurrence of Ca(2+)-induced Ca2+ release. When the caffeine-sensitive stores were emptied by prolonged application of caffeine, the amplitude and the rate of rise of the depolarization-induced [Ca2+]in transients were decreased. These facts suggest the involvement of internal caffeine-sensitive calcium stores in the generation of calcium signal in sensory neurons.

Different action of ethosuximide on low- and high-threshold calcium currents in rat sensory neurons.

The effects of ethosuximide on calcium channels were studied on dorsal root ganglion neurons from one-day-old rats using the patch-clamp technique. Bath application of ethosuximide induced dose-dependent and reversible suppression of calcium currents without affecting their time-course. Substantial differences between the effects of ethosuximide on the low-threshold and high-threshold (T- and L-) currents were observed. Ethosuximide reduced the T-current with greater potency than the L-current (Kd for T-current is 7 microM vs 15 microM for L-current). This relative specificity of its action still remained if applied at concentrations up to 1 mM. These data support the hypothesis according to which the anti-epileptic action of ethosuximide is related to reduction of the low-threshold calcium currents in sensory neurons.

Calcium signal prolongation in sensory neurones of mice with experimental diabetes.

Depolarization-induced Ca2+ transients were studied in dorsal root ganglion neurones of different size (large, 30-45 microns; small, 18-25 microns in diameter) from normal and diabetic mice. Whereas in large neurones no definite changes in the amplitude and time course of the transients were observed, in small neurones the decay of transient became substantially prolonged during streptozotocin-induced and spontaneously occurring diabetes. As small and large neurones differ substantially in their mechanisms of Ca2+ transient termination, we conclude that the prolongation of Ca2+ transients, probably induced by chronic hyperglycaemia, is specific only for small sensory neurones (transmitting mostly nociceptive signals) and may be a cause of the increased pain sensitivity often accompanying this disease.

[Lack of low-threshold calcium channels in the membrane of cultured cells of the heart ventricle of newborn rats]. / Otsutstvie nizkoporogovykh kal'tsievykh kanalov b membrane kul'tiviruemykh kletok zheludochka serdtsa novorozhdennykh krys.

In isolated, cultured neonatal rat ventricular myocytes calcium current was investigated by whole-cell patch clamp technique. The shift of holding potential values from -100-80 mV to -40 mV caused no significant changes in calcium current potential-dependent and kinetic properties. No calcium current component with preferential steady-state inactivation at membrane potentials ranged between -100 and -60 mV was observed. Calcium current component resistant to classic Ca-antagonists action was not found either. All data listed above permit a conclusion on lack of the low-threshold calcium channels in the membrane of single cultured neonatal rat ventricular myocytes. Possible reasons of the absence of this type of calcium are discussed.

[The characteristics of the sodium currents across EGTA-modified calcium channels in the membrane of cultured rat cardiomyocytes]. / Kharakteristika natrievykh tokov cherez EGTA-modifitsirovannye kal'tsievye kanaly membrany kul'tiviruemykh kardiomiotsitov krys.

In isolated, cultured neonatal rat ventricular myocytes sodium currents through calcium channels induced by lowering of extracellular calcium concentration 100 nmol/l have been investigated by whole-cell patch clamp technique. Such Na(+)-carried currents are modulated by classic Ca2+ agonists and antagonists. The potential-dependent characteristics of Na+ current are shifted at 20 mV in hyperpolarizing direction as compared to initial Ca(2+)-carried current. The inactivation decay of Na+ current through Ca2+ channels has the monoexponential behaviour. The possible action of extracellular Ca2+ lowering on Ca2+ channel selective filter and gating mechanisms is suggested.

[Effect of reduced extracellular level of sodium ions on the intracellular level of calcium ions in the cytoplasm of cultured rat cardiomyocytes]. / Vliianie snizheniia vnekletochnoi kontsentratsii ionov natriia na vnutrikletochnuiu kontsentratsiiu ionov ka'ltsiia v tsitoplazme ku'ltiviruemykh kardiomiotsitov krys.

Cytosolic free calcium [( Ca2+]in) was measured using fura-2 in isolated cultured ventricular myocytes of neonatal rat. Exposure of the cardiomyocyte to a solution in which all Na+ have been replaced by impermeable cations results in a 400-600 nmol/l increase of [Ca2+]in. This increase is followed by a slow decrease to the initial level. A decrease of the extracellular calcium concentration from 2.5 to 0.5 mmol./l or increase to 10 mmol/l produced, respectively, decrease and increase of the amplitude of [Ca2+]in rise in response to low-Na+ superfusion. Exposure of cardiomyocytes to low-Na+ solutions also led to a 2-3 fold increase of caffeine++-dependent Ca2+ release from intracellular stores. Changes in [Ca2+]in can be attributed to the operation of a sodium-calcium exchanger in heart cells.

[Use of a microfluorometric method for measuring free calcium in the cytoplasm of isolated cultured rat cardiomyocytes]. / Primenenie metoda mikrofliuorometrii dlia izmereniia svobodnogo ka'ltsiia v tsitoplazme odinochnykh ku'ltiviruemykh kardiomiotsitov krysy.

Dual wavelength microfluorometry was used to measure the cytoplasmic free calcium concentration [( Ca2+]in) in single cultured cells from ventricular myocytes of neonatal rats loaded with the indicator fura-2. At 2.5 nmol/l extracellular Ca2+ in the resting cells [Ca2+]in was between 80 and 110 nmol/l. Sometimes, spontaneous low-frequency (approximately 0.1 Hz) [Ca2+]in oscillations were observed. High-potassium depolarization led to a Ca2+-antagonists-sensitive rise of [Ca2+]in. Both caffeine++ (5-10 mmol/l) and thymol (lmmol/l) initialized transient increase of [Ca2+]in. Mechanisms of [Ca2+]in homeostasis in heart muscle cells were discussed.

Calcium currents in aged rat dorsal root ganglion neurones.

1. The whole-cell voltage clamp technique was used to record calcium currents in the somatic membrane of rat cultured dorsal root ganglion neurones. 2. Neurones were enzymatically isolated from animals of three age groups (neonatal, 2-7 days; adult, 7 months; and old, 30 months) and maintained in primary culture 3-14 days. 3. The neurones isolated from neonatal and old rats showed two distinct types of Ca2+ currents, a low-threshold transient current and a high-threshold sustained current, whereas neurones from old rats showed only a high-threshold calcium current. 4. The density of the high-threshold calcium current was 28.4 +/- 6.3 pA/pF (mean +/- S.E.M., n = 54) in neonatal, 39.1 +/- 7.2 pA/pF (n = 62) in adult and 11.0 +/- 4.6 pA/pF (n = 64) in old dorsal root ganglion neurones. 5. We found no difference in elementary high-threshold Ca2+ current characteristics in neurones from different age groups. The single-channel conductance was (with 60 mM Ca2+ in the recording pipette) 16.0 +/- 2.7 pS (mean +/- S.E.M., n = 9) in neonatal, 16.2 +/- 1.7 pS (n = 11) in adult and 16.4 +/- 1.2 pS (n = 12) in old neurones. 6. Current-voltage relations and kinetics of high-threshold calcium currents showed no detectable age-dependent difference. 7. The run-down of high-threshold calcium currents in dorsal root ganglion neurones from old rats was practically insensitive to intracellular administration of cyclic AMP and ATP. The same intervention caused a significant deceleration of Ca2+ current run-down in the majority of neonatal and in some adult cells. 8. We suggest that the disappearance of the low-threshold calcium current and reduction of high-threshold calcium current with ageing is due to a depression of calcium channel expression during late ontogenesis. The decrease of sensitivity of high-threshold calcium channels to phosphorylation by cyclic AMP-dependent protein kinase in aged neurones could also be a reason for altered turnover between silent and functional pools of calcium channels, which may underlie the age-dependent decline in the density of high-threshold calcium channels.