Xenon reduces glutamate-, AMPA-, and kainate-induced membrane currents in cortical neurones.

BACKGROUND: The anaesthetic, analgesic, and neuroprotective effects of xenon (Xe) are believed to be mediated by a block of the NMDA (N-methyl-D-aspartate) receptor channel. Interestingly, the clinical profile of the noble gas differs markedly from that of specific NMDA receptor antagonists. The aim of this study was, therefore, to investigate whether Xe might be less specific, also inhibiting the two other subtypes of glutamate receptor channels, such as the alpha-amino-3-hydroxy-5-methyl-4-isoxazolole propionate (AMPA) and kainate receptors. METHODS: The study was performed on voltage-clamped cortical neurones from embryonic mice and SH-SY5Y cells expressing GluR6 kainate receptors. Drugs were applied by a multi-barreled fast perfusion system. RESULTS: Xe, dissolved at approximately 3.45 mM in aqueous solution, diminished the peak and even more the plateau of AMPA and glutamate induced currents. At the control EC(50) value for AMPA (29 microM) these reductions were by about 40 and 56% and at 3 mM glutamate the reductions were by 45 and 66%, respectively. Currents activated at the control EC(50) value for kainate (57 microM) were inhibited by 42%. Likewise, Xe showed an inhibitory effect on kainate-induced membrane currents of SH-SY5Y cells transfected with the GluR6 subunit of the kainate receptor. Xe reduced kainate-induced currents by between 35 and 60%, depending on the kainate concentration. CONCLUSIONS: Xe blocks not only NMDA receptors, but also AMPA and kainate receptors in cortical neurones as well as GluR6-type receptors expressed in SH-SY5Y cells. Thus, Xe seems to be rather non-specific as a channel blocker and this may contribute to the analgesic and anaesthetic potency of Xe.

Gap junction communication and connexin 43 gene expression in a rat granulosa cell line: regulation by follicle-stimulating hormone.

Follicle-stimulating hormone is the major regulator of growth and development of antral follicles in the ovary. Granulosa cells (GCs) in these follicles are coupled via gap junctions (GJs) consisting of connexin 43 (Cx 43). Because we and others have found that Cx 43 and GJs, respectively, are more abundant in large antral follicles compared with small antral and preantral follicles, we hypothesized that FSH may control Cx 43 gene expression, GJ formation, and intercellular communication. To directly address these points, we chose a rat GC line (GFSHR-17) expressing the FSH receptor and the Cx 43 gene. The functionality of FSH receptors was shown by the effects of porcine FSH, namely cell rounding, reduced cellular proliferation, and stimulation of progesterone production of GFSHR-17 cells, which are effects that were detectable within hours. Treatment with FSH also statistically significantly increased Cx 43 mRNA levels, as shown after 6 to 9 h in Northern blots. These effects were antedated by altered GJ communication, which was observed within seconds. Using a single-cell/whole-cell patch clamp technique, we showed that FSH rapidly and reversibly enhanced electrical cell coupling of GFSHR-17 cells. Increased GJ communication was associated with statistically significantly decreased phosphorylation of Cx 43, which was observed within 10 min after FSH addition, during immunoprecipitation experiments. Our results demonstrate, to our knowledge for the first time, that the gonadotropin FSH acutely and directly stimulates intercellular communication of GFSHR-17 cells through existing GJs. Moreover, FSH also increases levels of Cx 43 mRNA. These changes are associated with reduced proliferation and enhanced differentiation of GFSHR-17 cells. In vivo factors in addition to FSH may be involved in the regulation of GJ/GJ communication between GCs in the follicle, but our results suggest that improved cell-to-cell coupling, enhanced Cx 43 gene expression, and possibly, formation of new GJs are direct consequences of FSH receptor activation and may antedate and/or initiate the pivotal effects of FSH on GCs.

Synaptosome-associated protein of 25 kilodaltons in oocytes and steroid-producing cells of rat and human ovary: molecular analysis and regulation by gonadotropins.

The synaptosome-associated protein of 25 kDa (SNAP-25) is crucially involved in exocytosis in neurons. The aim of this study was to investigate whether it is present in the ovary. We found SNAP-25 to be expressed in nonneuronal cells of the rat and human ovary, namely in all oocytes and in steroidogenic cells, including granulosa cells (GC) of large antral follicles and luteal cells. Both isoforms, SNAP-25a and b, were found in the ovary. Oocytes obtained by laser capture microdissection were shown to express SNAP-25b, whereas SNAP-25a was found in rat GC and human luteinized GC. Immunohistochemical observations of strong SNAP-25 staining in GC of large growing antral follicles compared with absent or weak staining in small follicles suggested a role in folliculogenesis. To study a presumed regulation of SNAP-25, we used a rat GC line (GFSHR-17), which expresses FSH receptors, and luteinizing human GC, which express LH receptors. FSH elevated SNAP-25 mRNA and protein levels about fivefold within 24 h in GFSHR-17 cells. The cAMP analogue dibutyryl-cAMP (db-cAMP) mimicked this action of FSH. The effects of both db-cAMP and FSH were inhibited by the protein kinase A (PKA) inhibitor H89. In contrast, SNAP-25 protein and mRNA-levels were not altered by LH/hCG in luteinized human GC. Our results for the first time identify SNAP-25b in oocytes and SNAP-25a in steroidogenic cells of the mammalian ovary. SNAP-25a and b may be involved in different exocytotic processes in these cell types.

Identification of an ovarian voltage-activated Na+-channel type: hints to involvement in luteolysis.

An endocrine type of voltage-activated sodium channel (eNaCh) was identified in the human ovary and human luteinized granulosa cells (GC). Whole-cell patch-clamp studies showed that the eNaCh in GC is functional and tetrodotoxin (TTX) sensitive. The luteotrophic hormone human CG (hCG) was found to decrease the peak amplitude of the sodium current within seconds. Treatment with hCG for 24-48 h suppressed not only eNaCh mRNA levels, but also mean Na+ peak currents and resting membrane potentials. An unexpected role for eNaChs in regulating cell morphology and function was indicated after pharmacological modulation of presumed eNaCh steady-state activity in GC cultures for 24-48 h using TTX (NaCh blocker) and veratridine (NaCh activator). TTX preserved a highly differentiated cellular phenotype. Veratridine not only increased the number of secondary lysosomes but also led to a significantly reduced progesterone production. Importantly, endocrine cells of the nonhuman primate corpus luteum (CL), which represent in vivo counterparts of luteinized GC, also contain eNaCh mRNA. Although the mechanism of channel activity under physiological conditions is not clear, it may include persistent Na+ currents. As observed in GC in culture, abundant secondary lysosomes were particularly evident in the regressing CL, suggesting a functional link between eNaCh activity and this form of cellular regression in vivo. Our results identify eNaCh in ovarian endocrine cells and demonstrate that their expression is under the inhibitory control of hCG. Activation of eNaChs in luteal cells, due to loss of gonadotropin support, may initiate a cascade of events leading to decreased CL function, a process that involves lysosomal activation and autophagy. These results imply that ovarian eNaChs are involved in the physiological demise of the temporary endocrine organ CL in the primate ovary during the menstrual cycle. Because commonly used drugs, including phenytoin, target NaChs, these results may be of clinical relevance.

Evidence for catecholaminergic, neuronlike cells in the adult human testis: changes associated with testicular pathologies.

Neuronlike, catecholaminergic cells expressing tyrosine-hydroxylase (TH) have recently been found in the testis of a nonhuman primate species, the rhesus monkey. We examined whether neuronlike cells are present in the human testis. To this end, we first determined if the genes for TH and for a voltage-activated sodium channel (NaCh), a prerequisite for neuronal excitability, are expressed in normal adult testes. Using an RT-PCR approach, cDNA clones, identical to the sequences of human TH and to the alpha subunit of a NaCh type, were isolated. Immunohistochemical methods localized the corresponding proteins in testicular biopsies from adult men (age range, 28-44 years) without testicular pathologies and from infertile patients with either Sertoli cell only (SCO) syndrome or severe hypospermatogenesis and germ cell arrest (GA). TH and NaCh antibodies, as well as antibodies recognizing dopamine-transporter protein, identified immunoreactive cells of mainly bipolar or occasionally multipolar, elongated phenotype in most, but not all, biopsies of each group (12 out of 23). The results were corroborated by identification of TH gene expression by RT-PCR approaches in biopsies. Immunoreactive cell bodies, as well as nerve fibers, were more readily detected in SCO and GA biopsies. This was quantified after immunohistochemically visualizing all testicular neuronal elements, cell bodies, and fibers, with a neurofilament 200 (NF-200) monoclonal antibody in one set of randomly selected sections from all biopsies. We found significantly increased NF-200-immunoreactive cell bodies and fibers in SCO-syndrome and GA biopsies. These results show the existence of an as yet unknown testicular catecholaminergic neuronlike cell type in the human testis. This cell type may complement and act in concert with the well-known testicular sympathetic innervation. The increase of both "intrinsic" (neuronal cells) and "extrinsic" (nerve fibers) neuronal elements in pathological testicular biopsies suggests that the two parts of the human testicular nervous system may be involved in pathogenesis and/or maintenance of GA and SCO syndromes.

Sources and function of neuronal signalling molecules in the gonads.

While the hypothalamic-pituitary-gonadal axis is crucial for the function of the gonads, non-endocrine regulatory influences are exerted by other factors within the gonads. Among these factors are neurotransmitters, such as catecholamines. Several types of receptors for catecholamines exist in the gonads on vascular or endocrine cells. Their activation can alter blood flow, steroidogenesis and gene expression, depending on the target cells. Recently a neuronal-like cell type expressing catecholamine-biosynthetic enzymes and neuronal proteins was identified in testis and ovary of human and non-human primates. Together with the well-known sympathetic innervation, this gonadal nervous system may serve as a source of catecholamines. Dopamine is present in the follicular fluid. Oocytes, while not able to perform de novo synthesis of catecholamines, were shown to utilize dopamine to produce norepinephrine. This catecholamine then acts on beta-adrenoreceptors of follicular cells to increase cAMP. Oocytes may thus indirectly via dopamine and cAMP be able to control their own meiotic arrest. In addition, neurotransmitters may also be synthesized in other, non-neuronal ovarian cells. Thus, cultured human granulosa-luteal cells possess the acetylcholine synthesizing enzyme and the acetylcholine-specific vesicular transporter protein. These cells also express muscarinic-receptors (M1), which are linked to the mobilization of intracellular calcium and cell proliferation. This suggests involvement of the acetylcholine system in follicular growth and in the periovulatory events. In neurons, neurotransmitters alter the properties of the neuronal cell membrane. If this is the case in endocrine cells of the gonads is not yet clear, but the recent identification of voltage-activated potassium and sodium channels in human luteinized granulosa-luteal cells raises this question and opens a door to a new area of investigation.