Respiration-related neurons in the medial nuclear complex of the solitary tract of the cat

Respiration-related (RR) neuronal activity was systematically searched for in the nuclear complex of the solitary tract (nTS) with special interest focused on regions located medially to the ventrolateral subnucleus (vlnTS) which is widely considered to be the anatomical location of the dorsal respiratory group (DRG). The experiments were carried out on cats under pentobarbitone anaesthesia, paralyzed and artificially ventilated. RR activity was recorded consistently not only in the vlnTS, but also in more medial regions. R alpha-type neurons were often encountered in ventral and medial aspects of the nTS. R beta-type neurons and expiration-related neurons were found also in ventral regions of the nTS. A small number of 'late onset' and 'early burst' inspiration-related neurons was found respectively in ventral and ventromedial regions of the nTS. The activity of neurons presenting a rhythmic pattern completely determined by lung volume changes due to the respirator was consistently recorded both in ventral and medial areas of the nTS. Most of these neurons were excited by lung inflation and displayed a discharge pattern characteristic of P-neurons. A few neurons, designated as 'inverse' P-neurons displayed an opposite behaviour, being inhibited by lung inflation. The results are consistent with both histological and neurophysiological studies showing that pulmonary stretch receptor afferents terminate also on the medial side of the solitary tract. It is suggested that the DRG is not restricted to the vlnTS, but comprises also neurons located in more medial regions of the nTS.     

Acetylcholine raises excitability by inhibiting the fast transient potassium current in cultured hippocampal neurons.

The effects of acetylcholine on cultured hippocampal neurons were investigated by using the whole-cell version of the patch-clamp technique. The CA1 region of the hippocampus was excised from brain slices of young rats (12-19 day old), incubated in a papain solution, and dissociated. Neurons were plated on a glial feeder layer. The experiments were conducted mostly on neurons cultured for 2-6 days. Upon depolarization under voltage clamp, these cells exhibited a fast transient outward current (A-current), which was inhibited by 4-aminopyridine (2.5 mM). Acetylcholine (0.1 microM) also inhibited this A-current, as did the muscarinic agonists bethanechol and muscarine. As expected from their inhibition of the A-current, acetylcholine and 4-aminopyridine both increased the amplitude of the action potential and prolonged its duration. We conclude that the inhibition of the A-current constitutes a mechanism by which acetylcholine exerts its excitatory influence on hippocampal neurons.     

Activation of a muscarinic receptor selectively inhibits a rapidly inactivated Ca2+ current in rat sympathetic neurons.

Sympathetic neurons dissociated from the superior cervical ganglion of 2-day-old rats were studied by whole-cell patch clamp and by fura-2 measurements of the cytosolic free Ca2+ concentration, [Ca2+]i. Step depolarizations in the presence of tetrodotoxin and hexamethonium triggered two Ca2+ currents that differed in the voltage dependence of activation and kinetics of inactivation. These currents resemble the L and N currents previously described in chicken sensory neurons [Nowycky, M. C., Fox, A. P. & Tsien, R. W. (1985) Nature (London) 316, 440-442]. Treatment with acetylcholine resulted in the rapid (within seconds), selective, and reversible inhibition of the rapidly inactivated, N-type current, whereas the long-lasting L-type current remained unaffected. The high sensitivity to blocker drugs (atropine, pirenzepine) indicated that this effect of acetylcholine was due to a muscarinic M1 receptor. Intracellular perfusion with nonhydrolyzable guanine nucleotide analogs or pretreatment of the neurons with pertussis toxin had profound effects on the Ca2+ current modulation. Guanosine 5'-[gamma-thio]triphosphate caused the disappearance of the N-type current (an effect akin to that of acetylcholine, but irreversible), whereas guanosine 5'-[beta-thio]diphosphate and pertussis toxin pretreatment prevented the acetylcholine-induced inhibition. In contrast, cAMP, applied intracellularly together with 3-isobutyl-1-methylxanthine, as well as activators and inhibitors of protein kinase C, were without effect. Acetylcholine caused shortening of action potentials in neurons treated with tetraethylammonium to partially block K+ channels. Moreover, when applied to neurons loaded with the fluorescent indicator fura-2, acetylcholine failed to appreciably modify [Ca2+]i at rest but caused a partial blunting of the initial [Ca2+]i peak induced by depolarization with high K+. This effect was blocked by muscarinic antagonists and pertussis toxin and was unaffected by protein kinase activators. Thus, muscarinic modulation of the N-type Ca2+ channels appears to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein and independent of both cAMP-dependent protein kinase and protein kinase C.     

A guanine nucleotide-binding protein mediates the inhibition of voltage-dependent calcium current by somatostatin in a pituitary cell line.

Somatostatin reduces voltage-dependent Ca2+ current (ICa) and intracellular free Ca2+ concentration in the AtT-20/D16-16 pituitary cell line. We tested whether guanine nucleotide-binding proteins (G or N proteins) are involved in the signal transduction mechanism between the somatostatin receptor and voltage-dependent Ca2+ channels. Treatment of the cells with pertussis toxin, which selectively ADP ribosylates the GTP binding proteins Gi and Go and suppresses the ability of Gi to couple inhibitory receptors to adenylate cyclase, abolished the action of somatostatin on both ICa and intracellular free Ca2+. Intracellular application of the nonhydrolyzable guanine nucleotide analog guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), which irreversibly activates G proteins, changed the somatostatin effect on ICa from a reversible to an irreversible inhibition. Intracellular GTP[gamma S] alone caused a very slowly developing inhibition of ICa. When ICa was inhibited by GTP[gamma S] (alone or with somatostatin), it failed to respond to subsequent applications of somatostatin. The effect of GTP[gamma S] on the inhibition of ICa by somatostatin was not altered by the intracellular application of cAMP and 3-isobutyl-1-methylxanthine. The results suggest that a GTP-binding protein is directly involved in the cAMP-independent receptor-mediated inhibition of voltage-dependent Ca2+ channels.     

cAMP-dependent, long-lasting inhibition of a K+ current in mammalian neurons.

We report the long-term modulation of K+ channels by cAMP in cultured murine colliculi neurons. A short (1-2 s) application of 8-Br-cAMP induced a long-lasting broadening of the action potential, a loss of after-hyperpolarization, and a reduction in spike accommodation. In agreement with these changes, 8-Br-cAMP produced a long-lasting (2 hr) inhibition of a K+ current. These effects were also observed after a short activation of the pituitary adenylyl cyclase-activating polypeptide, beta-adrenergic, and 5-hydroxytryptamine type 4 (5-HT4) receptors, all known to increase cAMP. A transient activation of the cAMP-dependent protein kinase and a long-lasting inhibition of phosphatases (up to 2 hr) were detected. The blockade of the K+ current resulting from a brief application of 8-Br-cAMP or 5-hydroxytryptamine was prolonged from 2 to 4 hr when protein-serine/threonine phosphatases 1 and 2A were inhibited with 10 nM okadaic acid. The critical steps following the cAMP-dependent protein kinase activation and resulting in a long-term blockade of phosphatases are discussed in this report.     

Age-related development of a heterozygous phenotype in solitary neurons of the homozygous Brattleboro rat.

A single-base deletion in the single-copy vasopressin gene is the cause of diabetes insipidus in the homozygous Brattleboro rat (di/di). It results in the synthesis of an altered vasopressin precursor of which the axonal transport is blocked. Paradoxically, a small number of solitary hypothalamic neurons displays all the immunoreactivities of the wild-type vasopressin precursor (i.e., vasopressin, neurophysin, and a glycopeptide). In the present paper we provide evidence that these neurons have undergone a switch to a genuine heterozygous (di/+) phenotype; i.e., they contain the immunoreactivities of both the wild-type and the mutated vasopressin precursors. In the neural lobe, glycopeptide fibers are also present, showing that axonal transport of the wild-type precursor is restored. Moreover, the number of neurons displaying this di/+ phenotype increases markedly and in a linear way (from 0.1% up to 3% of the vasopressin cells) with age. These findings indicate that after mitotic division has ceased, genomic alterations occur in somatic neurons in vivo. The molecular event generating the di/+ phenotype in the di/di animal could involve a somatic intrachromosomal gene conversion between the homologous exons of the vasopressin and the related oxytocin genes.     

Egg-laying hormone of Aplysia induces a voltage-dependent slow inward current carried by Na+ in an identified motoneuron.

This report presents studies on ionic currents in Aplysia motoneuron B16 that are modulated by the neuropeptide egg-laying hormone (ELH) of Aplysia. ELH induces an inward current that persists in the presence of the peptide and that decays slowly after ELH is removed from the bath. The effect is not due to a decrease in the delayed potassium current, the calcium-activated potassium current, or the transient potassium current. Current-voltage measurements indicate that ELH produces increased inward currents from -80 mV to approximately equal to 0 mV. The effect is particularly enhanced in the region from -40 mV to -25 mV where a negative slope conductance due to voltage-dependent slow inward current is observed. The slow inward current and the response to ELH persist in saline solutions in which Ca2+ is replaced with Co2+ but are eliminated when Na+ is replaced with equimolar concentrations of either Tris or N-methyl-D-glucamine. The response to ELH is unaffected by replacing chloride with equimolar acetate; by increasing the potassium concentration; or by adding tetraethylammonium chloride, CsCl, 4-amino-pyridine, or tetrodotoxin to the saline bath. In addition, the reversal potentials for the ELH response (range, -28 to +46 mV), obtained from difference current-voltage relationships, are consistent with an increase in the Na+-dependent slow inward current. We conclude that at least one of the effects of ELH on B16 is to increase a slow inward current carried by Na+.     

Somatostatin potentiates the alpha 1-adrenergic activation of phospholipase C in striatal astrocytes through a mechanism involving arachidonic acid and glutamate.

As previously shown with adenosine, somatostatin, which is ineffective alone, enhanced the alpha 1-adrenergic-agonist-stimulated production of inositol phosphates in cultured striatal astrocytes. This effect was suppressed in cells pretreated with pertussis toxin. It required external calcium and was selectively antagonized by both mepacrine, an inhibitor of phospholipase A2, and 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable analog of arachidonic acid. In addition, a long-lasting elevation of cytosolic calcium and a release of arachidonic acid were observed only under the combined stimulation of somatostatin and alpha 1-adrenergic receptors. Arachidonic acid could in turn inhibit glutamate uptake into astrocytes, and the resulting external accumulation of glutamate could account for the somatostatin-evoked amplification of the alpha 1-adrenergic-agonist-stimulated hydrolysis of inositol-phospholipids. The effect of somatostatin was indeed reproduced by glutamate or glutamate uptake inhibitors and suppressed by enzymatic removal of external glutamate. Thus, astrocytes may contribute to long-term plasticity events in glutamatergic synapses through regulation of external glutamate levels.     

Zinc modulation of a transient potassium current and histochemical localization of the metal in neurons of the suprachiasmatic nucleus.

The effect of Zn2+ on a voltage-dependent, transient potassium current (IA) in acutely dissociated neurons from the suprachiasmatic nucleus was studied with the whole-cell patch-clamp technique. At micromolar concentrations, Zn2+ markedly potentiated IA activated from a holding potential of -60 mV, which is the resting potential of these neurons. This potentiation occurred at a Zn2+ concentration as low as 2 microM and increased with higher Zn2+ concentrations. The Zn2+ action appears to arise from a shift in the steady-state inactivation of IA to more positive voltages. At 30 microM, Zn2+ shifted the half-inactivation voltage by +20 mV (from -80 mV to -60 mV), and 200 microM Zn2+ shifted this voltage by +45 mV (from -80 mV to -35 mV). Histochemically, we have also observed Zn2+ staining throughout the suprachiasmatic nucleus; the staining is particularly intense in the ventrolateral region of the nucleus, which receives the major fiber inputs. Our findings suggest that Zn2+, presumably synaptically released, may modulate the electrical activity of suprachiasmatic nucleus neurons through IA. Because vesicular Zn2+ is fairly widespread in the central nervous system, it is conceivable that this kind of Zn2+ modulation on IA, and possibly on other voltage-activated currents, exists elsewhere in the brain.     

Modulation of a transient K+ current in the pleural sensory neurons of Aplysia by serotonin and cAMP: implications for spike broadening.

To study the contribution of cAMP to the spike broadening produced by serotonin (5-HT) in the pleural sensory neurons of the tail withdrawal reflex, we utilized two phosphodiesterase-resistant cAMP analogs: the Sp diastereomer of cyclic adenosine 3',5'-monophosphothioate (Sp-cAMP[S]), which activates protein kinase A, and the antagonist Rp diastereomer of cyclic adenosine 3',5'-monophosphothioate (Rp-cAMP[S]), agonist Sp-cAMP[S] was injected into the sensory neurons, it caused spike broadening comparable to that induced by 5-HT. In turn, the cAMP antagonist Rp-cAMP[S] blocked approximately 50% of the 5-HT-induced spike broadening. We next examined the K+ currents that are modulated by 5-HT and determined how these currents are affected by cAMP. Confirming Baxter and Byrne [(1989) J. Neurophysiol. 62, 665-679], we found that 5-HT modulated two currents, an S-type K+ current (IKS) as well as a transient and voltage-dependent K+ current (IKV). Rp-cAMP[S] blocked the reduction by 5-HT of the early phase of IKV in parallel with, and to the same degree (60%), as this inhibitor blocked the IKS and spike broadening. These results support the idea that in the pleural sensory neurons cAMP mediates a significant part of the spike broadening that accompanies short-term facilitation produced by 5-HT and that cAMP can produce spike broadening by modulating both IKV and IKS.     

Tumor necrosis factor-alpha downregulates the voltage gated outward K+ current in cultured neonatal rat cardiomyocytes: a possible cause of electrical remodeling in diseased hearts.

BACKGROUND: Inflammatory cytokines have been reported to contribute to the progression of cardiac remodeling in various heart diseases and a remarkable prolongation of the monophasic action potential duration and reductions in the expression of Kv4.2 and K+ channel-interacting protein-2 (KChIP-2) in a rat autoimmune myocarditis model have been documented. In this study, the effect of tumor necrosis factor-alpha (TNF-alpha) on cultured cardiomyocytes was evaluated, focusing on the change in the voltage-gated outward K+ current and expression of related molecules. METHODS AND RESULTS: Cardiomyocytes isolated from 1-day-old Lewis rats were cultured for 72 h and treated with TNF-alpha (50 ng/ml) for an additional 48 h. The myocytes treated with TNF-alpha showed a 22% reduction in the peak K+ current, which consisted of a transient outward K+ current (Ito) and 1.4-fold enhancement of the cell-capacitance in comparison with the control. Among the cardiac ion channel related molecules evaluated in this study, Kv4.2 and KChIP-2 mRNA exhibited remarkable reductions (p < 0.05). CONCLUSIONS: Treatment with TNF-alpha induced reductions in Ito as well as cellular hypertrophy in neonatal cultured myocytes, which indicates that TNF-alpha might play a role in promoting electrical remodeling of cardiomyocytes under inflammatory conditions.     

Neurotransmitter release is blocked intracellularly by botulinum neurotoxin, and this requires uptake of both toxin polypeptides by a process mediated by the larger chain.

Botulinum neurotoxins (types A and B), which are microbial proteins consisting of two disulfide-linked chains, inhibit specifically and with high potency the release of acetylcholine from peripheral nerve terminals. As a prerequisite for a long-term development of effective treatments for botulism, the internalization and inhibitory action of the toxin and its constituent chains were examined by electrophysiological methods at identified synapses in Aplysia preparations that allow both intracellular and bath application of the neurotoxins. Intracellular recordings from cholinergic cells of the buccal ganglion demonstrated that extra- or intracellular application of low doses of botulinum neurotoxin results in a specific blockade of evoked transmitter release, without changing the quantal size; an intraneuronal site of action has thus been established. In contrast, release from noncholinergic neurons of cerebral ganglion was prevented by the neurotoxin only after injection into the cell. Purified preparations of the individual renatured chains, shown to be nontoxic in a mouse bioassay, failed to affect acetylcholine release when applied extra- or intracellularly. However, inhibition of release was observed after intracellular administration of both chains or when the light chain was injected and the heavy chain was bath-applied. These findings show that both chains are required on the cytosolic side of the neuronal plasma membrane for expression of toxicity and that the cholinergic specificity of the neurotoxin is attributable to its heavy chain, which mediates targeting and subsequent neuronal uptake.     

Stimulation of lipolysis in cultured fat cells by tumor necrosis factor, interleukin-1, and the interferons is blocked by inhibition of prostaglandin synthesis.

Multiple cytokines induce a number of alterations in lipid metabolism which can produce hyperlipidemia. Recent studies have demonstrated that tumor necrosis factor (TNF) increases lipolysis, resulting in an increase in circulating FFA levels, which stimulates hepatic triglyceride production, thereby contributing to the hyperlipidemia induced by TNF. In the present investigation we have determined the effects of a variety of cytokines on lipolysis in cultured 3T3-F442A adipocytes. TNF increased lipolysis approximately 3-fold with a maximal effect at 100 ng/ml and a half-maximal increase at 5-10 ng/ml. This increase was first observed 8 h after incubation with TNF. Interleukin-1 (IL-1) and interferon-alpha (IFN), -beta, and -gamma also stimulated lipolysis in cultured adipocytes. The half-maximal increase in lipolysis occurred at approximately 10 ng/ml IL-1, 5 ng/ml IFN alpha, 10 ng/ml IFN beta, and 8 ng/ml of IFN gamma. Maximal lipolysis was observed at approximately 100 ng/ml for each of these cytokines, with the exception of IFN beta, for which maximal stimulation was observed at 1000 ng/ml. Neither platelet-activating factor nor IL-6 stimulated lipolysis; therefore, it is unlikely that these compounds mediate the increase in lipolysis induced by cytokines. However, indomethacin, a well known inhibitor of prostaglandin synthesis, prevented the increase in lipolysis induced by TNF, IL-1, IFN alpha, IFN beta, or IFN gamma. Indomethacin did not affect basal lipolysis or the acute stimulation of lipolysis induced by epinephrine. These results demonstrate that multiple cytokines can increase lipolysis and that this increase is mediated by cytokine-induced stimulation of prostaglandin synthesis.     

Control of the light-regulated current in rod photoreceptors by cyclic GMP, calcium, and l-cis-diltiazem.

The effect of calcium ions on the cGMP-activated current of outer segment membrane was examined by the excised-patch technique. Changes in the extracellular calcium concentration had marked effects on the cGMP-activated current, while changes in intracellular calcium concentration were ineffective. Changes in calcium concentration in the absence of cGMP had little, if any, effect on membrane conductance. These results suggest that both intracellular cGMP and extracellular calcium can directly affect the conductance underlying the light response in rod cells. The pharmacological agent l-cis-diltiazem reversibly inhibited the cGMP-activated current when applied to the intracellular side of an excised patch. When superfused over intact rod cells, l-cis-diltiazem reversibly blocked much of the normal light response. The isomer, d-cis-diltiazem, did not significantly affect either patches or intact rod cells. Thus, the light-regulated conductance has binding sites for both calcium and cGMP that may interact during the normal light response in rod cells and a site specific for l-cis-diltiazem that can be used to identify and further study the conductance mechanism.     

Determination of the orientation of a DNA binding motif in a protein-DNA complex by photocrosslinking.

We have developed a straightforward biochemical method to determine the orientation of the DNA binding motif of a sequence-specific DNA binding protein relative to the DNA site in the protein-DNA complex. The method involves incorporation of a photoactivatable crosslinking agent at a single site within the DNA binding motif of the sequence-specific DNA binding protein, formation of the derivatized protein-DNA complex, UV-irradiation of the derivatized protein-DNA complex, and determination of the nucleotide(s) at which crosslinking occurs. We have applied the method to catabolite gene activator protein (CAP). We have constructed and analyzed two derivatives of CAP: one having a phenyl azide photoactivatable crosslinking agent at amino acid 2 of the helix-turn-helix motif of CAP, and one having a phenyl azide photoactivatable crosslinking agent at amino acid 10 of the helix-turn-helix motif of CAP. The results indicate that amino acid 2 of the helix-turn-helix motif is close to the top-strand nucleotides of base pairs 3 and 4 of the DNA half site in the CAP-DNA complex, and that amino acid 10 of the helix-turn-helix motif is close to the bottom-strand nucleotide of base pair 10 of the DNA half site in the CAP-DNA complex. The results define unambiguously the orientation of the helix-turn-helix motif relative to the DNA half site in the CAP-DNA complex. Comparison of the results to the crystallographic structure of the CAP-DNA complex [Schultz, S., Shields, S. & Steitz, T. (1991) Science 253, 1001-1007] indicates that the method provides accurate, high-resolution proximity and orientation information.     

Critical obstruction of the right ventricular outflow tract by a primary hemangioendothelioma in a seven month old.

A 7-month-old presented with failure to thrive and a murmur. Echocardiography demonstrated a large mass in the right ventricular outflow tract, extending through the pulmonary valve. During anaesthetic induction this caused critical obstruction of the outflow tract and cardiac arrest. Pathological diagnosis showed the lesion to be a primary hemangioendothelioma. Despite surgical excision and steroid therapy, the mass continued to grow for a period of 8 weeks, but then began to regress spontaneously.     

Exposure of platelet fibrinogen receptors by a monoclonal antibody to the GPIIb-IIIa complex, PMA4.

We found that a monoclonal antibody to the glycoprotein (GP) IIb-IIIa complex, PMA4, induces fibrinogen binding to platelets, and we examined the mechanism involved. Affinity chromatography and crossed immunoelectrophoresis showed that PMA4 recognized an epitope on the GPIIb-IIIa complex-specific domain. The binding of 125I-fibrinogen to platelets was induced by PMA4 in a concentration-dependent manner and was blocked by EDTA, RGDS peptides and an anti-GPIIb-IIIa monoclonal antibody, PMA1. Binding of the divalent antibody to the GPIIb-IIIa complex was necessary to induce fibrinogen binding and subsequent platelet aggregation, since Fab fragments, unlike PMA4 IgG and F(ab')2 fragments, did not induce fibrinogen binding or aggregation. The PMA4 IgG induced fibrinogen binding, serotonin secretion, and Ca2+ mobilization, whereas F(ab')2 induced fibrinogen binding only. In addition, F(ab')2-induced fibrinogen binding was not abolished in the presence of aspirin, H-7, a protein kinase C inhibitor, PGE1 or dibutyryl cyclic AMP. These results demonstrate that the binding of PMA4 divalent molecules to the GPIIb-IIIa complex can expose platelet fibrinogen receptors in the absence of the stimulatory effects of intracellular mediators on platelets. Thus, we conclude that the fibrinogen receptors on the GPIIb-IIIa complex can be exposed by direct action of the antibody on the complex molecules.     

Hormone replacement therapy in the geriatric patient: current state of the evidence and questions for the future. Estrogen, progesterone, testosterone, and thyroid hormone augmentation in geriatric clinical practice: part 1

This article presents an up-to-date review of the literature on hormone augmentation in the elderly to help primary care physicians better evaluate and utilize hormone replacement and optimization strategies to benefit their patients. The scientific literature suggests that hormone supplementation with estrogen, progesterone, testosterone, growth hormone, and thyroid hormone has the potential to improve quality of life and to prevent, or reverse, the many symptoms and conditions associated with aging, including fatigue, depression, weight gain,frailty, osteoporosis, loss of libido, and heart disease. Possible long-term side effects are also considered.