Inhibition of intracellular Ca2+ mobilisation by low antiproliferative concentrations of thapsigargin in human vascular smooth-muscle cells.

Low nanomolar concentrations of thapsigargin, a modulator of intracellular Ca2+ ([Ca2+]i) pools, inhibit vascular smooth-muscle cell (VSMC) proliferation. Because the mechanisms underlying this effect have not been defined, the effect of antiproliferative concentrations of thapsigargin on [Ca2+]i in fura-2-loaded VSMCs was studied by using dynamic video imaging of [Ca2+]i. After seeding on coverslips, human VSMCs were incubated for 1-48 h with thapsigargin before loading with fura-2 or during imaging. Mobilisation of [Ca2+]i was stimulated with 1 microM ionomycin in Ca2+-free medium and the increase in [Ca2+]i detected by using Ca2+ imaging techniques. Continuous exposure of cells to low concentrations of thapsigargin (which failed measurably to increase in [Ca2+]i) reduced the ionomycin response in a time- and dose-dependent manner (100% inhibition at 10 nM thapsigargin after 1 h exposure). After exposure of cells to 10 nM thapsigargin for 1 h followed by washing and further incubation for < or = 72 h, there was a time-dependent recovery of the ionomycin response. Because the concentrations of thapsigargin and exposure times are identical to those that inhibit replication in VSMCs, it is proposed that depletion of [Ca2+]i pools mediates the inhibitory effect of thapsigargin on VSMC proliferation.

The influence of surgical preparation on cyclic nucleotide synthesis in an organ culture of human saphenous vein.

OBJECTIVES: To investigate adenosine cyclic 3'5' monophosphate (cAMP) and guanosine cyclic 3'5' monophosphate (cGMP) synthesis in freshly isolated and surgically prepared human saphenous vein before and after culture. SETTING: Bristol Heart Institute, Bristol, U.K. METHODS: Freshly isolated and surgically prepared human saphenous vein was obtained from patients undergoing coronary artery bypass graft surgery. cAMP and cGMP synthesis, was assessed by radioimmunoassay in response to specific simulators in segments of saphenous veins after collection and following 14 days culture. RESULTS: Immediately after collection there was a significant reduction in the synthesis of cAMP (forskolin and prostaglandin E1-stimulated) and cGMP (sodium nitroprusside-stimulated) in surgically prepared compared to freshly isolated saphenous veins. In contrast, following 14 days in culture, cAMP and cGMP synthesis was significantly elevated in surgically prepared compared to freshly isolated saphenous veins. CONCLUSIONS: These data indicate that surgical preparation results in a marked reduction in cyclic nucleotide synthesis in saphenous vein which may be relevant to the pathophysiology of early vein graft failure. The normalisation of both cAMP and cGMP synthesis in surgically prepared veins following 14 days culture indicates that cyclic nucleotide synthesising capacity may not be a major determinant of neointima formation in this experimental model.

Reduced prostacyclin and increased leukotriene B4 synthesis in porcine venous-arterial grafts.

BACKGROUND: Migration and proliferation of vascular smooth muscle cells in the intima and superimposed atheroma are the main changes underlying late failure of saphenous vein bypass grafts. There is evidence that these events are partly modulated by complex interactions between inhibitors of vascular smooth muscle cell proliferation, such as prostacyclin (PGI2), and mitogens, such as leukotriene B4 (LTB4). Because the relative balance between these eicosanoids may play a role in vein graft failure, the synthesis of PGI2 and LTB4 was measured in porcine saphenous vein-carotid artery grafts 4 weeks after implantation and compared with ungrafted vein and common carotid artery from the same animal. METHODS: Vessels were cut into 2-mm squares and preincubated in Dulbecco's minimum essential medium for 4 hours at 37 degrees C. Tissues were then further incubated with Dulbecco's minimum essential medium containing a range of concentrations of noradrenaline, arachidonate, and calcium ionophore A23187. Release of PGI2 and LTB4 into the supernatant was then assessed by radioimmunoassay. RESULTS: In response to all stimulators, PGI2 release was markedly diminished in vein grafts compared with ungrafted saphenous veins and carotid arteries. The patterns of responses were similar in each vessel type. In contrast, LTB4 release was significantly enhanced in vein grafts compared to ungrafted saphenous veins and carotid arteries. CONCLUSIONS: These data indicate that there is a down-regulation of cyclooxygenase or PGI2 synthase in porcine vein grafts, which may constitute a further phenotypic change that would augment the hyperplastic process. Local increases in LTB4 synthesis in the vein graft, which indicates an induction of lipoxygenase and LTB4 synthase enzymes (and possibly reflects release from leukocytes which have infiltrated the graft), may contribute to increased intimal proliferation by direct promitogenic effects on smooth muscle cells.

The effects of idazoxan and other alpha 2-adrenoceptor antagonists on urine output in the rat.

1. In normally-hydrated Wistar rats the alpha 2-adrenoceptor antagonist, idazoxan (1, 3, 10 mg kg-1 i.p.), increased urine output during the 6 h following injection. 2. The more selective and specific alpha 2-adrenoceptor antagonist, RX811059 (0.3, 1, 3 mg kg-1 i.p.), and the peripherally-acting alpha 2-adrenoceptor antagonist, L-659,066 (1, 3, 10 mg kg-1 i.p.), had no effect on urine output in normally-hydrated animals. 3. In rats given a 25 ml kg-1 water-load orally, idazoxan (10 mg kg-1, i.p.) produced an initial antidiuretic response which was followed by an increase in urine output which was apparent 4 and 6 h after drug administration. 4. RX811059 (1, 3 mg kg-1 i.p.) and L-659,066 (3, 10 mg kg-1 i.p.) significantly decreased urine output in water-loaded rats in the 2 h after injection. 5. The antidiuretic effects of L-659,066 were attenuated in Brattleboro rats which are deficient in vasopressin; only the highest dose (10 mg kg-1 i.p.) decreased urine output, and this was only a small response in comparison with its virtual abolition of urine output in water-loaded Wistar rats. 6. The results with the selective alpha 2-adrenoceptor antagonists in Wistar and Brattleboro rats suggest that alpha 2-adrenoceptors in the periphery may play a physiological role in the control of water balance through a mechanism which involves vasopressin. 7. The paradoxical diuretic effects of idazoxan contrast with the effects of the other alpha 2-adrenoceptor antagonists and therefore may be attributed to a property of this compound unrelated to alpha 2-adrenoceptor blockade.

The vasopressin precursor in the Brattleboro (di/di) rat.

The vasopressin precursor in the rat hypothalamus has been studied, using trypsin to release desglycinamide vasopressin and coupling it to glycinamide (T & G treatment). The resulting amidated nonapeptide was detected and measured with a radioimmunoassay for vasopressin. The "vasopressin" produced in this way had the full immunoreactivity of the authentic peptide but eluted from an hplc column 1 min earlier and appeared to have a larger molecular weight. It was found that T&G treatment generated vasopressin immunoreactivity in extracts of the supraoptic nucleus (SON) of the Brattleboro rat in just the same way as it did in normal animals. Furthermore, this procedure produced vasopressin immunoreactivity in those hplc fractions from Brattleboro SON extracts that corresponded with the elution time of vasopressin precursor. Similar amounts of "vasopressin" could be generated from Brattleboro and normal SONs. These results support the suggestion that the Brattleboro SON synthesizes an aberrant vasopressin precursor which is not processed by the cell.

Two vasopressin-like peptides in the pig testis?

Vasopressin (VP)-like immunoreactivity (IR) has been located in the testes of several species of mammal. There is evidence that most of this IR in the rat does not represent authentic arginine vasopressin (AVP) and that a second AVP-like peptide may exist. We have studied testis samples from the pig, which produces lysine vasopressin (LVP) in its pituitary, and have found both LVP- and AVP-like IR. High-performance liquid chromatography (HPLC) of testis extracts showed two peaks of VP-IR. The first peak co-eluted with authentic LVP and was recognized only by antisera which cross-reacted with LVP. The second peak co-eluted with authentic AVP and was recognized by antisera raised against AVP. Both VP-like peptides bound to a neurophysin affinity column and the HPLC elution profiles of the bound peptides were similar to those of the authentic hormones. When the LVP-like material was oxidized with performic acid, a peak of IR running in the same position as oxidized authentic LVP on HPLC was produced. Similarly, the performic acid-oxidized AVP-like material co-eluted with oxidized authentic AVP. The presence of both LVP- and AVP-like peptides in the pig testis may mean that more than one gene is involved. A second VP-like gene could also explain the anomalies of VP-IR in other species.

Biosynthesis of oxytocin in the corpus luteum.

In this report we demonstrate that ovine and bovine luteal cells synthesise oxytocin by way of a precursor protein similar to that found in the hypothalamus. Isolated ovine or bovine luteal cells were incubated for up to 12 h with [35S]cysteine. Neurophysin-Sepharose column separation and HPLC of cell extracts demonstrated the presence of [35S]oxytocin. Incorporation of [35S]cysteine was confirmed by performic acid oxidation. Immunoprecipitation of cell extract with anti-rat oxytocin-neurophysin followed by SDS-PAGE yielded 2 radioactive bands of 14 kDa and 11-12 kDa. Immunoprecipitation with anti-oxytocin yielded 1 band at 14 kDa. On SDS-PAGE the 14 kDa band had a similar mobility to rat-hypothalamic oxytocin precursor.

Analysis of the neurohypophyseal components accumulating in the supraoptic nucleus of the rat after injection of colchicine.

[35S]Cysteine has been injected into the supraoptic nuclei of normal rats and of animals given 7 micrograms colchicine into the cerebrospinal fluid to inhibit transport of neurosecretory granules. Analysis of extracts of the supraoptic nuclei 20 min or 6 h after isotope injections showed that (i) colchicine does not affect biosynthesis, i.e., incorporation of tracer into the common precursors of neurohypophyseal hormones and their associated neurophysins, and (ii) processing of precursors inside the arrested granules proceeds normally.

Characterization of oxytocin, vasopressin, and neurophysin from the bovine corpus luteum.

Acid extracts of corpora lutea collected from nonpregnant cows were found to contain oxytocin, arginine vasopressin, and neurophysin. The inhibition curves of the oxytocin and vasopressin extracts showed parallelism with the appropriate standard preparations in specific RIAs and eluted at the same position as the standards using high performance liquid chromatography (HPLC). The neurophysin extract showed parallelism in a bovine neurophysin I RIA and had a similar elution position to the standard on both Sephadex G-50 and HPLC. However, its immunoreactive profile on HPLC differed slightly from that obtained with hypophyseal bovine neurophysin I. In nonpregnant cows the oxytocin content (about 1 microgram g-1 wet wt of tissue) was three orders of magnitude greater than the vasopressin content. Levels of luteal oxytocin were considerably lower in pregnant animals. These results show that the bovine ovary is a rich source of neurohypophysial peptides and suggest that oxytocin biosynthesis may occur within the corpus luteum.

Precursors in the biosynthesis of vasopressin and oxytocin in the rat. Characteristics of all the components in high-performance liquid chromatography.

A reverse-phase high performance liquid-chromatography (h.p.l.c.) protocol has been developed, whereby all the major known posterior-pituitary components that are derived from the processing of pro-oxytocin and pro-vasopressin can be separated one from another. Thus, in a single chromatographic step, it has been possible to separate vasopressin (VP), oxytocin (OT), oxytocin-neurophysin (rOT-Np), vasopressin-neurophysin (rVP-Np) and vasopressin-glycopeptide (rVP-GP) from acid extracts of the neurointermediate lobes of rat pituitary glands. All these peptides except rVP-GP were labelled in the neural lobe by 24h after a hypothalamic injection of [35S]cysteine, whereas all except VP were labelled by 24h after a similar injection of [3H]leucine. Three major labelled proteins were isolated from 20 min [35S]cysteine-injected rats when extracts of the supraoptic nucleus were subjected to Sephadex G-75 chromatography, h.p.l.c. and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Immunoprecipitation with antisera raised against rat neurophysins, VP and OT revealed 21000- and 19000-mol.wt. common precursors to VP and rVP-Np and a 15000-mol.wt. common precursor to OT and rOT-Np. Some immunoreactive rVP-Np could occasionally be detected in the Vo of Sephadex G-75 chromatograms of Wistar rat supraoptic-nucleus extracts, but no evidence of [35S]neurophysin in this fraction was obtained from h.p.l.c. fingerprinting of its S-carboxymethylated tryptic digests. Radioimmunoassay for rVP-Np and rOT-Np revealed that about 70-80% of the total recovered immunoreactive neurophysin (IR-Np) in the supraoptic nucleus eluted from Sephadex G-75 and h.p.l.c. in the positions of rVP-Np and rOT-Np. Evidence is presented for an approx. 20000-mol.wt. rOT-Np in both Wistar and Brattleboro rats and for an approx. 20000-mol.wt. component in the Brattleboro rat that is recognized by vasopressin-neurophysin antisera.

Effects of small doses of colchicine on the components of the hypothalamo-neurohypophysial system of the rat.

Small doses (3.5 micrograms and 7 micrograms) of colchicine injected intracisternally caused an interruption of transport of secretory material from the supraoptic and paraventricular nuclei of the hypothalamus to the neural lobe of the pituitary gland. Transport was assessed by direct measurement of the incorporation of [35S] cysteine into neurophysins, by radioimmunoassay of accumulated material in discrete areas of the system and by immunocytochemistry. The larger dose (7 micrograms) switched off transport completely during the first 24h but the system began to recover within three to four days. Colchicine had little, if any, effect on synthesis; comparison of the relationships of the apparent amounts of immunoreactive neurophysins and immunoreactive hormones in the arrested product led to the conclusion that processing of the hormone precursors continues within secretory granules which accumulated in the perikarya.