alpha-SNS produces the slow TTX-resistant sodium current in large cutaneous afferent DRG neurons

In this study, we used sensory neuron specific (SNS) sodium channel gene knockout (-/-) mice to ask whether SNS sodium channel produces the slow Na(+) current ("slow") in large (>40 microm diam) cutaneous afferent dorsal root ganglion (DRG) neurons. SNS wild-type (+/+) mice were used as controls. Retrograde Fluoro-Gold labeling permitted the definitive identification of cutaneous afferent neurons. Prepulse inactivation was used to separate the fast and slow Na(+) currents. Fifty-two percent of the large cutaneous afferent neurons isolated from SNS (+/+) mice expressed only fast-inactivating Na(+) currents ("fast"), and 48% expressed both fast and slow Na(+) currents. The fast and slow current densities were 0.90 +/- 0.12 and 0.39 +/- 0.16 nA/pF, respectively. Fast Na(+) currents were blocked completely by 300 nM tetrodotoxin (TTX), while slow Na(+) currents were resistant to 300 nM TTX, confirming that the slow Na(+) currents observed in large cutaneous DRG neurons are TTX-resistant (TTX-R). Slow Na(+) currents could not be detected in large cutaneous afferent neurons from SNS (-/-) mice; these cells expressed only fast Na(+) current, and it was blocked by 300 nM TTX. The fast Na(+) current density in SNS (-/-) neurons was 1.47 +/- 0. 14 nA/pF, approximately 60% higher than the current density observed in SNS (+/+) mice (P < 0.02). A low-voltage-activated TTX-R Na(+) current ("persistent") observed in small C-type neurons is not present in large cutaneous afferent neurons from either SNS (+/+) or SNS (-/-) mice. These results show that the slow TTX-R Na(+) current in large cutaneous afferent DRG is produced by the SNS sodium channel.     

Role of nitric oxide in the biology, physiology and pathophysiology of reproduction

Following its benchmark discovery, nitric oxide (NO) is nowknown to play important functional roles in a variety of physiologicalsystems. Within the vasculature, NO induces vasodilation, inhibitsplatelet aggregation, prevents neutrophil/platelet adhesionto endothelial cells, inhibits smooth muscle cell proliferationand migration, regulates programmed cell death (apoptosis) andmaintains endothelial cell barrier function. NO generated byneurons acts as a neurotransmitter, whereas NO generated bymacrophages in response to invading microbes acts as an antimicrobialagent. Because neurons, blood vessels and cells of the immunesystem are integral parts of the reproductive organs, and inview of the important functional role that NO plays in thosesystems, it is likely that NO is an important regulator of thebiology and physiology of the reproductive system. Indeed, inthe past 10 years, NO has established itself as a polyvalentmolecule which plays a decisive role in regulating multiplefunctions within the female as well as the male reproductivesystem. This review provides an overview of the role of NO invarious reproductive organs under physiological and pathologicalconditions.     

ATRX encodes a novel member of the SNF2 family of proteins: mutations point to a common mechanism underlying the ATR-X syndrome

It was shown recently that mutations of the ATRX gene give rise to a severe, X-linked form of syndromal mental retardation associated with alpha thalassaemia (ATR-X syndrome). In this study, we have characterised the full-length cDNA and predicted structure of the ATRX protein. Comparative analysis shows that it is an entirely new member of the SNF2 subgroup of a superfamily of proteins with similar ATPase and helicase domains. ATRX probably acts as a regulator of gene expression. Definition of its genomic structure enabled us to identify four novel splicing defects by screening 52 affected individuals. Correlation between these and previously identified mutations with variations in the ATR-X phenotype provides insights into the pathophysiology of this disease and the normal role of the ATRX protein in vivo.      

Long-term effects of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione in the rat basal ganglia: calcification, changes in glutamate receptors and glial reactions

Previous data from our laboratory indicate that 25 mM ibotenic acid induces intracellular calcifications in the rat basal forebrain. Because of the lack of specificity of ibotenic acid for a glutamate receptor subtype, a dose-response study with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate was undertaken and calcified areas (identified with Alizarin Red staining) as well as astro- and microglial reactions (by autoradiography with [3H]lazabemide and [3H]Ro 5-4864) were quantified at one month post-lesion. alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionate administered into the globus pallidus induced, in a dose-dependent manner, the formation of calcium deposits and the activation of both glial cells, the microglial reaction being particularly robust. From this study, a dose of 5.4 mM alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate was selected for further experiments. [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate, [3H]dizocilpine maleate and [3H]PN 200-110 binding in vitro were performed to assess autoradiographically whether the tissue damage was associated with changes in glutamate receptors and calcium channel binding sites. In the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-treated animals, the specific binding of [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate was significantly reduced by 28% in the lesioned ventral pallidum, whereas it was unchanged in the globus pallidus and substantia innominata. In these three nuclei, calcifications developed and an increase in both glial markers was measured. In contrast, the binding of [3H]PN 200-110 and [3H]dizocilpine maleate were unaffected. Co-injection of 15 mM 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione, a selective alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptor antagonist, prevented the formation of calcium concretions, the microglial reaction and the decrease in [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate binding but it failed to inhibit totally the astroglial reaction induced by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate. This may suggest that the microglial reaction and calcification take place through different mechanisms from the astrogliosis associated with the neuronal loss. In conclusion, acute administration of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate in the rat globus pallidus elicits a dose-dependent calcification process associated with a chronic reaction of astrocytes and microglia. alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-induced injury is accompanied by a slight reduction of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors in the ventral pallidum, whereas the binding of N-methyl-D-aspartate and L-type calcium channels receptors remains unchanged in any lesioned nucleus.     

Assessment of bioequivalence after subcutaneous and intramuscular administration of urinary gonadotrophins

The objective was to demonstrate bioequivalence between s.c. and i.m. administration of Humegon (FSH/LH ratio 1:1) and Normegon (FSH/LH ratio 3:1). In two randomized, single-centre, cross-over studies, 18 healthy volunteers on each formulation were assigned to one of the two administration sequences. Subjects were given single doses of one of the above gonadotrophins after endogenous gonadotrophin production had first been suppressed using high-dose oral contraceptive. Subsequently, rate (Cmax, tmax) and extent (AUC) of absorption of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were determined for 14 days. For Cmax and AUC, analysis of variance (ANOVA) was performed on log-transformed data and for tmax ANOVA was performed on ranks. Intramuscular and s.c. injections of Humegon were bioequivalent with respect to the main pharmacokinetic parameters, being AUC and Cmax of FSH absorption. Intramuscular and s.c. injections of Normegon were bioequivalent with respect to the AUC of FSH and not bioequivalent with respect to the Cmax of FSH. For tmax of FSH as well as for most LH variables of both preparations, bioequivalence could not be proven due to the high intra- and interindividual variability and/or concentrations being close to the detection limit. Thus, the main pharmacokinetic FSH variables after i.m. and s.c. administration of Humegon and Normegon were bioequivalent.      

Relationship between dose of methotrexate, apoptosis, p53/p21 expression and intestinal crypt proliferation in the rat

Abstract Previous studies have shown that apoptosis is induced by cytotoxic chemotherapy and precedes hypoproliferation of intestinal crypt cells. However, the relationship between the degree of intestinal apoptosis and crypt cell hypoproliferation may not be directly related. The purpose of this study was to investigate the relationship between apoptosis and hypoproliferation with increasing doses of chemotherapy. Eleven groups of breast cancer-bearing DA rats were treated with two doses of methotrexate (MTX) i. m. at varying concentrations (0.5, 1.5, 2.5 and 5.0 mg/kg) or saline (control). Animals were killed at 6 or 24 h following treatment. The small and large intestines were examined for apoptosis, villous area (small intestine), crypt length and mitotic count per crypt. Immunohistochemical expression of p53 and p21^waf1/cip1 (p21) were examined quantitatively. Data were analysed using Peritz F-test. Low dose MTX (0.5 mg/kg) did not change p53 expression at 6 h but induced a 15-fold increase in apoptosis in the crypts of the small intestine. This was associated with only a minor reduction in crypt cell proliferation. Higher doses of MTX increased p53 expression and caused a lower (7-fold) but more prolonged peak of apoptosis that was accompanied by reduced villous area, shortened crypts and a more profound reduction in crypt cell proliferation. Unlike the small intestine, apoptosis in the colon was 10-fold lower, proportional to the dose of MTX and did not induce overt damage. Expression of p21 did not change with any dose at either timepoint. We conclude that apoptosis is not always associated with crypt cell hypoproliferation and that the small intestine can recover after low dose MTX despite a heightened peak of apoptosis of crypt cells.     

Membrane ionic currents and properties of freshly dissociated rat brainstem neurons

It is well known that neuronal firing properties are determined by synaptic inputs and inherent membrane functions such as specific ionic currents. To characterize the ionic currents of brainstem cardio-respiratory neurons, cells from the hypoglossal (XII) nucleus and the dorsal motor nucleus of the vagus (DMX) were freshly dissociated and membrane ionic currents were studied under whole-cell voltage and current clamp. Both of these neurons showed a TTX-sensitive Na⁺ current with a much larger current density in XII than DMX neurons. This Na⁺ current had two (fast and slow) distinct inactivation decay components. The ratio of the magnitudes of the fast to slow component was roughly two-fold greater in DMX than in XII cells. Both DMX and XII neurons also showed a high voltage-activated Ca²⁺ current, but this current density was significantly greater (three-fold) in DMX than XII neurons. A relatively small amount of low-voltage activated Ca²⁺ current was also observed in DMX neurons, but not in the majority of XII cells. A transient and a sustained outward current components were observed in DMX cells, but only sustained currents were present in XII neurons. These outward currents had a reversal potential of about − 70 mV with 3 mM external K⁺ and −30 mV with 25 mM K⁺, and substitution of K⁺ with cesium and tetraethylammonium suppressed more than 90% the outward currents, indicating that most outward currents were carried by K⁺. The transient outward current consisted of two components with onesensitive to 4-aminopyridine and the other to intracellular Ca²⁺. In XII neurons, BRL 38227 (lemakalim), an ATP-sensitive K⁺ (K_ATP) channel activator, increased the sustained K⁺ currents by 10% of control, and glibenclamide, a K_ATP channel blocker, decreased the sustained K⁺ currents by 20%. Evidence for the presence of an inward rectifier K⁺ current was also obtained from both XII and DMX neurons. These results on XII and DMX neurons indicate that (1) the methods used to dissociate neurons provide a useful means to overcome voltage clamp technical difficulties; (2) ion channel characteristics such as density and biophysical properties of DMX neurons are very different from those of XII neurons; and (3) several newly discovered membrane ionic currents are present in these cells.