Calcium current characterization in dissociated adult guinea-pig jugular ganglion neurons.

Voltage-dependent calcium (Ca2+) channel currents in freshly dissociated adult guinea-pig jugular ganglion neurons were examined and characterized using the whole-cell patch-clamp technique. Electrophysiological analysis demonstrated a high-threshold current, but no low-threshold or T-type current. A fraction of the total Ca2+ current was inhibited by omega (omega)-conotoxin GVIA (Cg (inTX; 10 microM); the dihydropyridine antagonist nifedipine (NIF; 10 microM), inhibited a large fraction of the CgTX-insensitive current. The remaining CgTX/NIF-insensitive current was completely inhibited by omega-agatoxin IVA (AgIVA; 100 nM). These results demonstrated that the whole-cell Ca2+ channel current consisted only of N-, L- P-type components. Of these currents, only the L-type was partially inhibited by both histamine and carbachol (0.01-100 microM).

Neurosteroids modulate calcium currents in hippocampal CA1 neurons via a pertussis toxin-sensitive G-protein-coupled mechanism.

The inhibition of Ca2+ channel currents by endogenous brain steroids was examined in freshly dissociated pyramidal neurons from the adult guinea pig hippocampal CA1 region. The steady-state inhibition of the peak Ca2+ channel current evoked by depolarizing steps from -80 to -10 mV occurred in a concentration-dependent manner with the following IC50 values: pregnenolone sulfate (PES), 11 nM; pregnenolone (PE), 130 nM; and allotetrahydrocorticosterone (THCC), 298 nM. THCC, PE, and PES depressed a fraction of the Ca2+ channel current with a maximal inhibition of 60% of the total current. However, substitution of an acetate group for the sulfate group on PES resulted in a complete loss of activity. Progesterone had no effect (4% inhibition at 100 microM). Intracellular dialysis of PES had no effect on the Ca2+ current; concomitant extracellular perfusion of PES showed normal inhibitory activity, suggesting that the steroid binding site can only be accessed extracellularly. Analysis of tail currents at -80 mV demonstrated that THCC and PES slowed the rate of Ca2+ current activation and deactivation with no change in the voltage dependence of activation. Inhibition of the Ca2+ channel current by THCC and PES was voltage dependent. THCC primarily inhibits the omega-conotoxin (CgTX)-sensitive or N-type Ca2+ channel current. PE was nonselective in inhibiting both the CgTX- and the nifedipine (NIF)-sensitive Ca2+ channel current. These neurosteroids had no effect on the CgTX/NIF-insensitive current. In neurons isolated from pertussis toxin (PTX)-treated animals by chronic intracerebroventricular infusion (1000 ng/24 hr for 48 hr), the Ca2+ channel current inhibition by PES, PE, and THCC was significantly diminished. Intracellular dialysis with GDP-beta-S (500 microM) also significantly diminished the neurosteroid inhibition of the Ca2+ channel current. Intracellular dialysis with the general kinase inhibitors H-7 (100 microM), staurosporine (400 nM), and a 20 amino acid protein kinase inhibitor (1 microM) also significantly prevented the THCC and PES inhibition of the Ca2+ channel current. Intracellular dialysis with the more specific inhibitors of protein kinase C (PKC), the pseudosubstrate inhibitor (PKCI 19-36) (1-2 microM) and bisindolylmaleimide (1 microM) significantly diminished the THCC and PE inhibition of the Ca2+ channel current. Rp- cAMP (100 microM), a specific inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the THCC and PE inhibition of the Ca2+ current.(ABSTRACT TRUNCATED AT 400 WORDS)

Muscarine modulation by a G-protein alpha-subunit of delayed rectifier K+ current in rat ventromedial hypothalamic neurones.

1. Rat cultured ventromedial hypothalamic (VMH) neurones obtained from embryonic hypothalamus were used to study the muscarinic (carbachol) modulation of voltage-gated K+ currents with the whole-cell patch-clamp technique. 2. Carbachol produced a potent and concentration-dependent (100 fM to 100 microM) decrease of the outward delayed rectifier K+ current (IK) with an IC50 of 44 pM and a Hill coefficient of 0.4. The carbachol-induced depression of IK was reduced by pirenzepine (1-10 microM) and atropine (1 microM). Carbachol had no effect on the transient outward K+ current (IA). 3. Intracellular dialysis with guanosine 5'-O-(2-thiodiophosphate) (GDP-beta-S, 500 microM) significantly diminished the carbachol-induced depression of IK, suggesting GTP-binding protein (G-protein) involvement. Pre-incubation of VMH neurones with pertussis toxin (200-400 ng ml-1) or cholera toxin (1 microgram ml-1) for 24-48 h had no effect on the carbachol-induced depression of IK. This suggested that the G alpha o, G alpha i, and G alpha s G-protein alpha-subunits were not involved in mediating the carbachol-induced depression of IK in VMH neurones. 4. Treatment (24-48 h) of VMH neurones with antisense phosphothio-oligodeoxynucleotides to the G alpha 11 G-protein subunit (10 microM) significantly diminished the carbachol-induced depression of IK. Treatment with 10 microM of either G alpha 11 sense or antisense to G alpha q had no effect. 5. These results demonstrate a novel and potent muscarinic depression of IK in VMN neurones, and that this depression is specifically mediated by the G alpha 11 G-protein subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterisation of the human lung NK-1 receptor cDNA.

Functional cDNA clones for human NK-1 receptor were isolated from human lung RNA using the polymerase chain reaction (PCR). We have screened a human cosmid library and isolated a clone which appeared to contain the entire NK-1 receptor gene. From the published rat NK-1 receptor cDNA sequence we designed primers within the protein coding sequence, but outwards towards both the 5' and 3' ends of the putative human protein sequence. By this method we derived DNA sequence from the 3' end of the human gene. In order to determine the 5' end of the gene we used a PCR based method called Rapid Amplification of cDNA Ends (RACE). From the derived human sequences amplimers were designed upstream of the ATG initiation codon and downstream of the stop codon. The entire cDNA was obtained by RNA-PCR from human lung RNA. The sequence obtained was 407 amino acids in length, encoding an open-reading frame that was highly homologous to the rat NK-1 receptor cDNA (89%). The entire human cDNA was then cloned into a mammalian expression vector and mRNA was synthesized by in vitro transcription. Applications of tachykinins caused membrane current responses in Xenopus oocytes injected with the in vitro synthesized mRNA. The most potent of the three tachykinin peptides tested was Substance P. The human NK-1 receptor gene has been mapped to chromosome 2 using the polymerase chain reaction to specifically amplify the human sequence in hamster/human hybrid DNA and also in mouse/human monochromosome hybrids.

Isolation and characterisation of the human lung NK-2 receptor gene using rapid amplification of cDNA ends.

Functional cDNA clones for human NK-2 receptor were isolated from human lung RNA using a polymerase chain reaction (PCR) based method (RACE-PCR). In this method the cDNA was isolated as 5' end and 3'-end fragments; the entire cDNA was obtained by RNA-PCR. The sequence derived was 398 amino acids in length encoding an open-reading frame that was highly homologous to both the bovine and rat NK-2 receptor. The entire human cDNA sequence was cloned into a mammalian expression vector and mRNA was synthesised by in vitro transcription. Applications of tachykinins caused membrane current responses in Xenopus oocytes injected with the in vitro synthesised mRNA. The most potent of the three tachykinin peptides tested was neurokinin A. We have screened a human cosmid library and isolated a clone which contains the entire NK-2 receptor gene. The gene contains five exons and we have determined the complete sequence of the exons and the intron-exon junctions.

Pentoxifylline, a methylxanthine derivative, prevents postsurgical adhesion reformation in rabbits.

Previous studies from our laboratory have demonstrated pentoxifylline to be a potent inhibitor of primary post-traumatic adhesion formation in a rodent model. To evaluate pentoxifylline in a situation more closely mimicking the events encountered in infertility surgery, we developed a model for adhesion reformation after lysis of pelvic adhesions. New Zealand White rabbits received a standardized primary traumatic lesion to the left uterine horn. One week later, a laparotomy was performed for evaluation (prescore) and subsequent lysis of adhesions. After closure, the animals were randomized to treatment with vehicle or subcutaneous pentoxifylline, 2.5 mg/kg, administered at 12-hour intervals for six doses. Seven days later, the rabbits were sacrificed and evaluated in a blinded manner to quantify adhesion reformation (postscore). Using a scoring scale from 0 = no adhesions to 4+ = most severe, the mean prescore was not different between pentoxifylline-treated and control rabbits (3.8 versus 3.9, respectively). However, the mean postscore (0.7 versus 3.7, respectively) was markedly reduced by pentoxifylline (P less than .001). These data demonstrate a marked inhibition of adhesion reformation after lysis of pelvic adhesions under the influence of pentoxifylline in rabbits.

Peritoneal fluid from endometriosis patients affects reproductive outcome in an in vivo model.

The cause and importance of endometriosis-associated subfertility are a subject of dispute in reproductive endocrinology. To further study this phenomenon, we have established a model to test the effect of peritoneal fluid (PF) from endometriosis patients on early reproductive events in vivo. Sexually mature female golden hamsters were subjected to an ovarian hyperstimulation protocol and divided into groups that received the following intraperitoneal injections: (1) saline, (2) human serum albumin (HSA), (3) PF from fertile controls, and (4) PF from stage I/II endometriosis patients. Animals were killed on days 4 and 17; reproductive performance was assessed by the number of oocytes and embryos recovered on day 4 and the number of uterine swellings counted on day 17. Reproductive performance was significantly impaired by PF from endometriosis patients; animals treated with control PF and HSA did not differ from control. These data demonstrate a marked impairment of early in vivo reproductive performance under the influence of endometriosis PF. These results support a role for a soluble PF component as a mediator in the pathogenesis of endometriosis-associated subfertility.