Thyroid hormones modulate thyrotropin-releasing hormone biosynthesis in tissues outside the hypothalamic-pituitary axis of male rats

In the present study we have examined the in vivo effects of thyroid hormone and TRH on secretory tissue concentrations of TRH and TRH-Gly (pGlu-His-Pro-Gly), a TRH precursor. Within secretory granules, TRH-Gly is converted to TRH through alpha-amidation of the C-terminal proline residue, using Gly as the NH2 donor. Using specific RIA, we measured the TRH-Gly immunoreactivity (TRH-Gly-IR) and TRH-IR concentrations in tissues from the reproductive and gastrointestinal systems, adrenals, and other internal organs in euthyroid, hypothyroid, and T4-treated 250-g Sprague-Dawley male rats. TRH-Gly-IR concentrations were more than 2-fold higher than TRH-IR concentrations within the adrenal, pancreas, bowel, and stomach at the time of death. Untreated hypothyroidism and exogenous TRH significantly increased adrenal TRH-Gly-IR levels. Pancreatic TRH-Gly levels increased about 2-fold in hypothyroid rats. Incubation at 60 C significantly increased TRH-Gly-IR levels in the pancreas, adrenal, bowel, stomach, and epididymis by 14-, 3-, 6-, 6-, and 6-fold, respectively. Also after 60 C incubation increases in the TRH-Gly-IR/TRH-IR ratio of 2.7-, 4-, and 1.7-fold were observed in the pancreas, epididymis, and bowel, respectively. Pooled tissue extracts were fractionated by cation exchange and reverse phase HPLC for characterization of TRH-Gly-IR. Both chromatographic methods revealed a major peak of TRH-Gly-IR coeluting with synthetic TRH-Gly. Incubation at 60 C caused 13.5-, 4.1-, 1.5-, and 5-fold increments in the TRH-Gly-IR for adrenal, pancreas, prostate, and thyroid, respectively, compared to the immediately extracted control aliquots. Cation exchange and reverse phase HPLC also revealed production of higher mol wt TRH precursor peptides after incubation at 60 C for 4 or 20 h. Only the TRH-Gly-IR peak coeluting with pGlu-His-Pro-Gly was converted into TRH by rat brain alpha-amidating enzyme. The data suggest that biosynthesis of TRH occurs in rat extrahypothalamic tissues and may be modulated by thyroid status, iv TRH, and selective thermal inactivation of enzymes that convert prepro-TRH to TRH.     

Effects of external pH on ionic currents in smooth muscle cells from the basilar artery of the guinea pig.

pHo is an important determinant of vascular tone in cerebral blood vessels. We investigated the effects of changes in pHo on isolated smooth muscle cells from the basilar artery of the guinea pig. Single cells contracted rapidly in response to an elevation in pHo (constant CO2), and contraction was blocked by nifedipine, suggesting a role for dihydropyridine-sensitive Ca2+ channels. In whole-cell patch-clamp experiments, changes in pHo (pHo 5.7-8.1, pHi 7.2 with 10 mM HEPES) strongly affected the amplitude of the peak Ca2+ channel current (10 mM Ba2+, +15 mV, holding potential of -55 mV), with an apparent pK of 6.9. The current-voltage curves were minimally shifted, indicating no important effect of surface charge. To separate the slowly inactivating L-type Ca2+ channel current from the more rapidly inactivating B-type current, the decaying portions of inward currents from cells studied with repetitive 1-second pulses (+15 mV, holding potential of -55 mV) were fit to a two-component model. Titration curves for the L-type and B-type currents indicated maximum increases by factors of 3.65 and 1.28 at alkaline pHo and gave apparent pK values of 7.71 and 6.47 (Hill coefficient unity). The time constant of inactivation for the B-type current at +15 mV was little affected by pHo, whereas that for the L-type current increased somewhat with increasing pHo. Additional experiments showed no significant effect of pHo on holding current or on voltage-activated outward currents (pCai 7 with 11 mM EGTA). Our results provide additional evidence for participation of Ca2+ channels in regulating basal tone in cerebral smooth muscle and indicate that pHo regulates current through slowly inactivating, dihydropyridine-sensitive L-type Ca2+ channels.     

Genetic epidemiology of cystic fibrosis in Saguenay-Lac-St-Jean (Quebec, Canada)

Cystic fibrosis (CF) is an autosomal recessive disorder with a prevalence at birth estimated at 1/2000-1/2500 livebirths in Caucasian populations. Some 127 CF individuals are known in Saguenay-Lac-St-Jean (SLSJ), a geographically isolated region of Quebec. The prevalence at birth was estimated at 1/902 live borns, and the carrier rate was estimated at 1/15 inhabitants in the SLSJ region. The mean inbreeding coefficient was only slightly elevated in the CF group compared with three control groups, and was due to remote consanguinity. The mean kinship coefficient was 2.4 times higher in the CF group than in the control groups. In SLSJ region, the places of origin of the CF individuals and their parents did not show a clustered nonuniform distribution. Endogamy was not higher in the CF group than in control groups.     

Functional integrity of endothelium determines Ca2+ channel availability in smooth muscle: involvement of nitric oxide

Endothelium regulates smooth muscle contractility in part via nitric oxide (NO). We tested the hypothesis that endothelial dysfunction, either produced by injury or simulated pharmacologically by reducing the bioavailability of NO, results in elevated Ca2+ channel availability (ngmax=maximum conductance/cell capacitance) in smooth muscle cells isolated from the vessel. Using basilar arteries of normotensive Wistar rats, we measured ngmax in smooth muscle cells from control vessels, from vessels in which endothelium was injured using Na fluoroscene plus light, and from vessels in which the bioavailability of NO was reduced by pretreatment with the NO scavenger 1H-imidazol-1 -yloxy,2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-3-oxide , potassium salt (C-PTIO), or the endothelial nitric oxide synthase (eNOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Values of ngmax in these four groups of cells were 0.28+/-0.02 nS/pF (n=22), 0.51+/-0.05 nS/pF (n=15), 0.430+/-.03 nS/pF (n=12), and 0.47+/-0.04 nS/pF (n=14) (P<0.05, ANOVA), respectively. To determine whether larger currents associated with endothelial dysfunction exhibit altered sensitivity to exogenous NO, we quantified the response to various concentrations of NO donor, Na nitroprusside (SNP), in 37 cells from control vessels and 33 cells from vessels pretreated with L-NAME. SNP exhibited identical potency (half-maximum values, 18.7 and 21.1 nM) but greater apparent efficacy (maximum fractional block, 0.82 versus 0.63) in down-regulating Ca2+ channel currents in cells isolated from vessels with dysfunctional endothelium. Our results are consistent with a direct influence of endogenous NO on Ca2+ channel availability in smooth muscle cells, and indicate that Ca2+ channel availability in isolated smooth muscle cells may be a sensitive measure of the functional integrity of the endothelium in the parent vessel.     

Comparison of caprine,human and bovine strains of respiratory syncytial virus

Summary A new continuous ovine kidney cell line allowing the growth of caprine, human and bovine respiratory syncytial virus was used to minimize host cell related variations for the direct comparison of the viral ultrastructures, serological relationships and structural protein profiles. Results show that all three strains are closely related although a closer relationship was found between bovine and caprine RS.     

Sodium nitroprusside and cGMP decrease Ca2+ channel availability in basilar artery smooth muscle cells

 We studied the effect of the nitric oxide (NO) donor, sodium nitroprusside (SNP), on the macroscopic and single-channel currents due to the 22-pS Ca²⁺ channel in smooth muscle cells from guinea pig basilar artery. In nystatin-perforated whole-cell recordings, 50 nM SNP decreased the macroscopic current to 63±12% of control values, without changing the voltage dependence of the current. In cell-attached patches with BAY-K8644 in the pipette, SNP caused a comparable decrease in single-channel availability (n ·P _o) that was dose dependent over the range of 10 nM to 10 μM SNP. SNP had no effect on single-channel properties, including slope conductance, voltage dependence of activation, the number of open states, the time constants of the open states, and the proportion of time spent in each open state. The effect of SNP (50 nM) on single Ca²⁺ channel openings was reproduced by 8-Br-cGMP (100 μM), which also reduced channel availability without altering channel properties. The protein kinase inhibitor H-8 (1.5 μM), which exhibits relative specificity for cGMP-dependent protein kinase, completely inhibited the decrease in single-channel availability expected with SNP. The dose-dependent decrease in Ca²⁺ channel availability caused by SNP was not altered by prior application of 8-Br-cAMP or forskolin, both of which cause an increase in Ca²⁺ channel availability in these cells. Our findings suggest that NO decreases openings of Ca²⁺ channels in basilar artery smooth muscle cells without altering channel properties, and that it does so by a mechanism likely to involve cGMP-dependent protein kinase. Received: 2 July 1996 / Received after revision: 30 September 1996 / Accepted: 2 October 1996     

Linkage disequilibrium analysis of childhood-onset spinal muscular atrophy (SMA) in the French -- Canadian population

Spinal muscular atrophy (SMA) is, after Duchenne muscular dystrophy,the most common neuromuscular disorder in childhood. The generesponsible for childhood SMA has been mapped to the q11. 2– q13. 3 region of chromosome 5. We have extended ourlinkage studies of SMA In the French - Canadian population toInclude microsatellite markers at the D5S125, D5S351, D5S435,JK53CA1/ 2 and MAPI B locl. These markers span about 4 cM ofthe SMA candidate region. We observed significant evidence forlinkage between SMA and all the markers tested. The analysisof recombinant chromosomes provide evidence for the followinggenetic order: D5S125-D5S435-MAP1B-3'-JK53CA1/2 and places D5S351proximal to JK53CA1/2. Furthermore, we confirm the current localizationof the SMA gene distal to D5S435. Finally, we provide demonstrationof significant linkage disequilibrium between childhood-onsetSMA and four of the five marker loci, D5S125, D5S435, D5S351and JK53CA1/2. Analysis of SMA-region haplotypes suggests thatthere may be a predominant SMA allele that is present on about17% of SMA chromosomes in this sample of the French - Canadianpopulation. We conclude that the observed linkage disequilibriumis likely due to genetic drift among regions of Quebec, consistentwith this population's early history.     

Role of the nuclear matrix in adenovirus maturation

The nuclear matrix has been implicated in several important cellular processes. In this paper, we investigate the role of the nuclear matrix in adenovirus type 2 assembly. Electron microscopic examination of nuclear matrices isolated from adenovirus infected Hep-2 cells clearly reveals that late in the lytic cycle, adenovirus capsids are intimately associated with the nuclear matrix. SDS-PAGE analysis showed that the viral core polypeptides V, PVII and 11 kDa were enriched in the nuclear matrix fraction. After a 3 h chase period a constant high ratio of PVII to VII prevailed in the nuclear matrix suggesting that mostly young virions and viral cores are bound to this structure. Most of the virus maturation endoproteinase activity co-purified with the nuclear matrix and the data suggest that the enzyme may be released from fragile young virions or assembly intermediates. Together these experiments suggest that the nuclear matrix is the site of adenovirus assembly and that mature virions may be released from the matrix by the viral endoproteinase.