Male contraception via simultaneous knockout of α1A-adrenoceptors and P2X1-purinoceptors in mice.

Therapeutic targets for male contraception are associated with numerous problems due to their focus on disrupting spermatogenesis or hormonal mechanisms to produce dysfunctional sperm. Here we describe the dual genetic deletion of α1A-adrenergic G protein-coupled receptors (adrenoceptors) and P2X1-purinoceptor ligand gated ion channels in male mice, thereby blocking sympathetically mediated sperm transport through the vas deferens during the emission phase of ejaculation. This modification produced 100% infertility without effects on sexual behavior or function. Sperm taken from the cauda epididymides of double knockout mice were microscopically normal and motile. Furthermore, double knockout sperm were capable of producing normal offspring following intracytoplasmic sperm injection into wild-type ova and implantation of the fertilized eggs into foster mothers. Blood pressure and baroreflex function was reduced in double knockout mice, but no more than single knockout of α1A-adrenoceptors alone. These results suggest that this autonomic method of male contraception appears free of major physiological and behavioral side effects. In addition, they provide conclusive proof of concept that pharmacological antagonism of the P2X1-purinoceptor and α1A-adrenoceptor provides a safe and effective therapeutic target for a nonhormonal, readily reversible male contraceptive.

Angiotensin type 1A receptors in C1 neurons of the rostral ventrolateral medulla modulate the pressor response to aversive stress.

The rise in blood pressure during an acute aversive stress has been suggested to involve activation of angiotensin type 1A receptors (AT(1A)Rs) at various sites within the brain, including the rostral ventrolateral medulla. In this study we examine the involvement of AT(1A)Rs associated with a subclass of sympathetic premotor neurons of the rostral ventrolateral medulla, the C1 neurons. The distribution of putative AT(1A)R-expressing cells was mapped throughout the brains of three transgenic mice with a bacterial artificial chromosome-expressing green fluorescent protein under the control of the AT(1A)R promoter. The overall distribution correlated with that of the AT(1A)Rs mapped by other methods and demonstrated that the majority of C1 neurons express the AT(1A)R. Cre-recombinase expression in C1 neurons of AT(1A)R-floxed mice enabled demonstration that the pressor response to microinjection of angiotensin II into the rostral ventrolateral medulla is dependent upon expression of the AT(1A)R in these neurons. Lentiviral-induced expression of wild-type AT(1A)Rs in C1 neurons of global AT(1A)R knock-out mice, implanted with radiotelemeter devices for recording blood pressure, modulated the pressor response to aversive stress. During prolonged cage-switch stress, expression of AT(1A)Rs in C1 neurons induced a greater sustained pressor response when compared to the control viral-injected group (22 ± 4 mmHg for AT(1A)R vs 10 ± 1 mmHg for GFP; p < 0.001), which was restored toward that of the wild-type group (28 ± 2 mmHg). This study demonstrates that AT(1A)R expression by C1 neurons is essential for the pressor response to angiotensin II and that this pathway plays an important role in the pressor response to aversive stress.

Baroreceptor reflex control of heart rate in angiotensin type 1A receptor knockout mice.

The baroreceptor reflex dampens the short-term fluctuations in blood pressure by feedback modulation of heart rate (HR) and vascular resistance. Impairment of this reflex has been observed in hypertension and heart failure. Angiotensin II, a blood borne hormone, acts via its type 1A receptor to attenuate the baroreceptor reflex and this reflex is reported to be dramatically altered in angiotensin type 1A receptor knockout mice. This study sought to further investigate changes in the arterial and cardiopulmonary baroreceptor reflex control of HR in angiotensin II type 1A receptor knocked out mice. In artificially ventilated, isoflurane anesthetized mice, the arterial and cardiopulmonary baroreceptor reflexes were activated via injection or slow infusions, respectively, of phenylephrine and sodium nitroprusside through the jugular vein. We observed no impairment of either the arterial or cardiopulmonary baroreceptor reflex control of HR in angiotensin type 1A receptor knockout mice.

Central angiotensinergic mechanisms associated with hypertension.

Following its generation by both systemic and tissue-based renin-angiotensin systems, angiotensin II interacts with specific, G-protein coupled receptors to modulate multiple physiological systems, including the cardiovascular system. Genetic models in which the different components of the renin-angiotensin system have been deleted show large changes in resting blood pressure. Interruption of the generation of angiotensin II, or its interaction with these receptors, decreases blood pressure in hypertensive humans and experimental animal models of hypertension. Whilst the interaction of angiotensin II with the kidney is pivotal in this modulation of blood pressure, an involvement of the system in other tissues is important. Both systemic angiotensins, acting via the blood-brain barrier deficient circumventricular organs, and centrally-generated angiotensin modulate cardiovascular control by regulating fluid and electrolyte ingestion, autonomic activity and neuroendocrine function. This review discusses the pathways in the brain that are involved in this regulation of blood pressure as well as examining the sites in which altered angiotensin function might contribute to the development and maintenance of high blood pressure.

Stimulation of angiotensin type 1A receptors on catecholaminergic cells contributes to angiotensin-dependent hypertension.

Hypertension contributes to multiple forms of cardiovascular disease and thus morbidity and mortality. The mechanisms inducing hypertension remain unclear although the involvement of homeostatic systems, such as the renin-angiotensin and sympathetic nervous systems, is established. A pivotal role of the angiotensin type 1 receptor in the proximal tubule of the kidney for the development of experimental hypertension is established. Yet, other systems are involved. This study tests whether the expression of angiotensin type 1A receptors in catecholaminergic cells contributes to hypertension development. Using a Cre-lox approach, we deleted the angiotensin type 1A receptor from all catecholaminergic cells. This deletion did not alter basal metabolism or blood pressure but delayed the onset of angiotensin-dependent hypertension and reduced the maximal response. Cardiac hypertrophy was also reduced. The knockout mice showed attenuated activation of the sympathetic nervous system during angiotensin II infusion as measured by spectral analysis of the blood pressure. Increased reactive oxygen species production was observed in forebrain regions, including the subfornical organ, of the knockout mouse but was markedly reduced in the rostral ventrolateral medulla. These studies demonstrate that stimulation of the angiotensin type 1A receptor on catecholaminergic cells is required for the full development of angiotensin-dependent hypertension and support an important role for the sympathetic nervous system in this model.

Cardiovascular role of angiotensin type1A receptors in the nucleus of the solitary tract of mice.

AIMS: The nucleus of the solitary tract (NTS) is important for cardiovascular regulation and contains angiotensin type 1A (AT1A) receptors. To assess its function, we examined the effect of expressing in AT1A receptors in the NTS of mice lacking these receptors. METHODS AND RESULTS: Bilateral microinjections of lentivirus expressing AT1A receptors (AT1Av mice, n = 6) or green fluorescent protein (GFPv, n = 8, control) under the control of the PRSx8 promotor were made into the NTS of AT1A receptors null mice (AT1A(-/-)). Telemetry devices recorded blood pressure (BP), heart rate (HR), and locomotor activity. Expression of AT1A receptors in the NTS increased BP by 11.2 ± 4 mmHg (P < 0.05) at 2 and 3 weeks, whereas GFPv mice remained at pre-injection BP. Ganglion blockade reduced BP to similar levels pre- and post-transfection in GFPv and AT1Av mice. Greater pressor responses to cage-switch stress were observed following AT1A receptors expression (+18 ± 2 mmHg pre- to +24 ± 2 mmHg post-virus, P < 0.05) with similar stress-induced pressor responses pre- and post-virus in GFPv mice. Pressor responses to restraint stress pre- and post-virus were similar in AT1Av but were 20% less post-GFPv (P < 0.001). The lack of attenuation in BP to restraint was associated with four-fold greater Fos-expression in AT1A receptors mice. AT1A receptors expression in the NTS did not alter baroreflex gain differently between groups. CONCLUSION: The results suggest that transfection of AT1A receptors on neurons in the NTS elevates BP independent of the SNS and pressor responses to aversive stimuli are associated with greater Fos-expression in forebrain regions. This study suggests a novel mechanism by which the NTS may modulate MAP in the long-term via AT1A receptors.