The in vitro pharmacological profile of KR31080, a nonpeptide AT1 receptor antagonist

Summary— KR31080 (2-butyl-5-methyl-6-(1-oxopyridin-2-yl)-3-[[2'-(1H-tetrazol-5-yl) biphenyl-4-yl]methyl]-3H-imidazo[4,5-b] pyridine) is a potent inhibitor of angiotensin type 1 (AT₁) receptors in rabbit aorta and human recombinant AT₁ receptors. In the isolated rabbit thoracic aorta, KR31080 caused a nonparallel shift to the right of the concentration-response curves to angiotensin II (All) with decreased maximal response (pD'₂ = 10.1 ± 0.1), but had no effect on the contractile response induced by norepinephrine. KR31080 inhibited specific [¹²⁵I]AII binding to rabbit aortic membranes (AT, receptors) and [¹²⁵I][Sar¹, Ile⁸]AII binding to human recombinant AT₁ receptors in a concentration-dependent manner with IC₅₀ values of 0.84 ± 0.08 nM and 1.92 ± 0.15 nM, respectively, but did not inhibit specific [¹²⁵I)AII binding to bovine cerebellum membranes (ÀT₂ receptors). In the Scatchard analysis, KR31080 interacted with rabbit aortic AT₁ receptors in a competitive manner, similar to losartan. These results demonstrate that KR31080 is a potent and AT₁ selective angiotensin receptor antagonist which exerts a competitive antagonism in the [¹²⁵I]AII binding assay and insurmountable AT₁ receptor antagonism in the functional study.     

Modulating role of dopamine on anesthetic requirements

The influence of dopamine on halothane anesthetic requirements was determined in mice. Halothane anesthetic requirement was defined as the minimum anesthetic concentration (MAC) that prevented 50% animals from moving in response to a supramaximal stimulus. Levodopa (L-DOPA) dose-dependently decreased halothane MAC to a maximum of 49% of control; over the same dose range L-DOPA increased striatal dopamine nearly 4-fold. The MAC-reducing effect of L-DOPA was attenuated by selective antagonism of the D2 dopamine receptor with YM-09151-2 while selective blockade of the D1 dopamine receptor with SCH-23390 did not alter L-DOPA's effect on the MAC for halothane. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) decreased striatal dopamine by 82% and increased the MAC for halothane by 27%. Repletion of striatal dopamine with L-DOPA, in MPTP-treated mice, restored the MAC for halothane back to the control state. The regression line derived from the plot of halothane MAC versus striatal dopamine content shows a highly significant correlation between the two variables (r2 = 0.94). These are the first results to suggest that anesthetic requirements can be modulated directly and precisely by increasing or decreasing the content of a single neurotransmitter in the central nervous system.     

Silicone-Coated Polypropylene Hollow-Fiber Oxygenator: Experimental Evaluation and Preliminary Clinical Use

Background. A membrane oxygenator consisting of a microporous polypropylene hollow fiber with a 0.2-μm ultrathin silicone layer (cyclosiloxane) was developed. Animal experimental and preliminary clinical studies evaluated its reliability in bypass procedures.

Methods. Five 24-hour venoarterial bypass periods were conducted on dogs using the oxygenator (group A). In 5 controls, bypass periods were conducted using the same oxygenator without silicone coating (group B). As a preliminary clinical study, 14 patients underwent cardiopulmonary bypass with the silicone-coated oxygenator.

Results. Eight to 16 hours (mean, 12.2 hours) after initiation of bypass, plasma leakage occurred in all group B animals, but none in group A. The O₂ and CO₂ transfer rates after 24 hours in group A were significantly higher than at termination of bypass in group B (p < 0.005 and p < 0.03, respectively). Scanning electron microscopy of silicone-coated fibers after 24 hours of bypass revealed no damage to the silicone coating of the polypropylene hollow fibers. In the clinical study, the oxygenator showed good gas transfer, acceptable pressure loss, low hemolysis, and good durability.

Conclusions. This oxygenator is more durable and offers greater gas transfer capabilities than the previous generation of oxygenators.     

LIPOPROTEIN(α) IN HEALTH AND DISEASE

SUMMARY Elevated plasma levels of Lp(a) do seem to influence the progression of atherosclerosis. Evidence is emerging that certain apo(a) isoforms may be more atherogenic than others, and in transgenic mice free apo(a) has been shown to be associated with accelerated atherosclerosis. Currently it is not known whether treating elevated Lp(a) levels will reduce progression of atherosclerosis and, as therapeutic options are limited, mass screening of Lp(a) levels in populations is not indicated. The presence of raised Lp(a) levels, however, warrants aggressive treatment to reduce other cardiovascular risk factors. Continuing research to investigate the relationship of the apo(a) gene to other genes, including the plasminogen gene and apo(a)-related genes, will add further information pertaining to the evolution, function, regulation and clinical implications of Lp(a).     

Role of the sympathetic nervous system in the maintenance of hypertension in rats harboring pheochromocytoma

Hypertension due to pheochromocytoma is generally considered to be a straightforward, direct consequence of the elevated concentrations of circulating catecholamines. However, clonidine, a centrally acting antihypertensive drug, has been reported to lower blood pressure in patients with pheochromocytoma, suggesting the possibility that the sympathetic nervous system is involved in the maintenance of hypertension in this disease. We have investigated this possibility in New England Deaconess Hospital rats harboring a transplantable pheochromocytoma that secretes norepinephrine and dopamine. Both clonidine and chlorisondamine, a ganglionic blocker, markedly decreased blood pressure in tumor-bearing rats. However, in other rats made acutely hypertensive with a norepinephrine infusion, neither clonidine nor chlorisondamine decreased blood pressure. This result indicates that in an acute model of hypertension, where baroreflex mechanisms have likely withdrawn sympathetic tone, neither clonidine nor chlorisondamine had nonspecific antihypertensive effects. A central nervous system site of action for the antihypertensive effect of clonidine in the rats harboring pheochromocytoma was suggested by the observation that the opiate antagonist naloxone both reversed and prevented clonidine's effect on blood pressure. Prazosin and yohimbine were utilized to determine the respective contributions of alpha-1 and alpha-2 adrenergic receptors in the maintenance of hypertension in rats harboring pheochromocytoma. Both drugs markedly lowered blood pressure in these rats. Our data suggest that both the sympathetic nervous system and circulating catecholamines are involved in the maintenance of hypertension due to pheochromocytoma.     

Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi- probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.      

Sequencing of the alpha-synuclein gene in a large series of cases of familial Parkinson's disease fails to reveal any further mutations. The European Consortium on Genetic Susceptibility in Parkinson's Disease (GSPD)

A mutation in exon 4 of the human alpha-synuclein gene was reported recently in four families with autosomal dominant Parkinson's disease (PD). In order to examine whether mutations in this exon or elsewhere in the gene are common in familial PD, all seven exons of the alpha- synuclein gene were amplified by PCR from index cases of 30 European and American Caucasian kindreds affected with autosomal dominant PD. Each product was sequenced directly and examined for mutations in the open reading frame. No mutations were found in any of the samples examined. We conclude that the A53T change described in the alpha- synuclein gene is a rare cause of PD or may even be a rare variant. Mutations in the regulatory or intronic regions of the gene were not excluded by this study.      

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.