Voiding behavior in awake unrestrained untethered spontaneously hypertensive and Wistar control rats.

The spontaneously hypertensive rat (SHR), a genetic model of high blood pressure, has also been studied as a potential model of overactive bladder. In vivo studies have confirmed the presence of surrogate markers of overactive bladder, including detrusor overactivity, increased urinary frequency, decreased bladder capacity and voided volume (VV), and afferent hypersensitivity to bladder irritation. However, these observations were during awake cystometry using implanted bladder catheters tethered to an infusion pump and artificially filled. We conducted experiments in awake unrestrained untethered age-matched female SHRs and Wistar rats to quantify naïve consumption and voiding behavior and the effect of capsaicin desensitization on consumption and voiding behavior. Food and water consumption, body weight, voiding frequency, and VV were recorded. Rats were placed in metabolism cages for 24 h, up to twice a week, from 17 to 37 wk of age. Compared with Wistar rats, SHRs exhibited decrease in VV and did not exhibit diurnal variation in VV between light and dark periods, suggesting that SHRs may have bladder hypersensitivity. Furthermore, SHRs may also have smaller bladder capacities, as they consumed less water, voided less volume (regardless of light cycle), and had equal urinary frequencies compared with age-matched Wistar rats. We detected no change in SHR voiding behavior following capsaicin desensitization, which was in contrast to a prior awake in vivo cystometry study describing increased VV and micturition interval in SHRs and suggests that C-fiber activity may not contribute to bladder hypersensitivity in SHRs.NEW & NOTEWORTHY We characterized the long-term (20 wk) voiding, defecation, and consumption behavior of age-matched spontaneously hypertensive and Wistar rats without the influence of anesthesia or catheters. Spontaneously hypertensive rats exhibited bladder hypersensitiviy that persisted for the 20-wk duration and was unaffected by capsacin desensitization.

Effects of intravesical prostaglandin E2 on bladder function are preserved in capsaicin-desensitized rats.

Prostaglandin E2 (PGE2) instilled into the bladder generates symptoms of urinary urgency in healthy women and reduces bladder capacity and urethral pressure in both humans and female rats. Systemic capsaicin desensitization, which causes degeneration of C-fibers, prevented PGE2-mediated reductions in bladder capacity, suggesting that PGE2 acts as an irritant (Maggi CA, Giuliani S, Conte B, Furio M, Santicioli P, Meli P, Gragnani L, Meli A. Eur J Pharmacol 145: 105-112, 1988). In the present study, we instilled PGE2 in female rats after capsaicin desensitization but without the hypogastric nerve transection that was conducted in the Maggi et al. study. One week after capsaicin injection (125 mg/kg sc), rats underwent cystometric and urethral perfusion testing under urethane anesthesia with saline and 100 µM PGE2. Similar to naïve rats, capsaicin-desensitized rats exhibited a reduction in bladder capacity from 1.23 ± 0.08 mL to 0.70 ± 0.10 mL (P = 0.002, n = 9), a reduction in urethral perfusion pressure from 19.3 ± 2.1 cmH2O to 10.9 ± 1.2 cmH2O (P = 0.004, n = 9), and a reduction in bladder compliance from 0.13 ± 0.020 mL/cmH2O to 0.090 ± 0.014 mL/cmH2O (P = 0.011, n = 9). Thus, changes in bladder function following the instillation of PGE2 were not dependent on capsaicin-sensitive pathways. Further, these results suggest that urethral relaxation/weakness and/or increased detrusor pressure as a result of decreased compliance may contribute to urinary urgency and highlight potential targets for new therapies for overactive bladder.

Chronic Electrical Stimulation of the Superior Laryngeal Nerve in the Rat: A Potential Therapeutic Approach for Postmenopausal Osteoporosis.

Electrical stimulation of myelinated afferent fibers of the superior laryngeal nerve (SLN) facilitates calcitonin secretion from the thyroid gland in anesthetized rats. In this study, we aimed to quantify the electrical SLN stimulation-induced systemic calcitonin release in conscious rats and to then clarify effects of chronic SLN stimulation on bone mineral density (BMD) in a rat ovariectomized disease model of osteoporosis. Cuff electrodes were implanted bilaterally on SLNs and after two weeks recovery were stimulated (0.5 ms, 90 microampere) repetitively at 40 Hz for 8 min. Immunoreactive calcitonin release was initially measured and quantified in systemic venous blood plasma samples from conscious healthy rats. For chronic SLN stimulation, stimuli were applied intermittently for 3-4 weeks, starting at five weeks after ovariectomy (OVX). After the end of the stimulation period, BMD of the femur and tibia was measured. SLN stimulation increased plasma immunoreactive calcitonin concentration by 13.3 ± 17.3 pg/mL (mean ± SD). BMD in proximal metaphysis of tibia (p = 0.0324) and in distal metaphysis of femur (p = 0.0510) in chronically SLN-stimulated rats was 4-5% higher than that in sham rats. Our findings demonstrate chronic electrical stimulation of the SLNs produced enhanced calcitonin release from the thyroid gland and partially improved bone loss in OVX rats.

Evaluating Associations Between Nonclinical Cardiovascular Functional Endpoints and Repeat-dose Cardiovascular Toxicity in the Beagle Dog: A Cross-company Initiative.

Integrating nonclinical in vitro, in silico, and in vivo datasets holistically can improve hazard characterization and risk assessment. In pharmaceutical development, cardiovascular liabilities are a leading cause of compound attrition. Prior to clinical studies, functional cardiovascular data are generated in single-dose safety pharmacology telemetry studies, with structural pathology data obtained from repeat-dose toxicology studies with limited concurrent functional endpoints, eg, electrocardiogram via jacketed telemetry. Relationships between datasets remain largely undetermined. To address this gap, a cross-pharma collaboration collated functional and structural data from 135 compounds. Retrospective functional data were collected from good laboratory practice conscious dog safety pharmacology studies: effects defined as hemodynamic blood pressure or heart rate changes. Morphologic pathology findings (mainly degeneration, vacuolation, inflammation) from related toxicology studies in the dog (3-91 days repeat-dosing) were reviewed, harmonized, and location categorized: cardiac muscle (myocardium, epicardium, endocardium, unspecified), atrioventricular/aortic valves, blood vessels. The prevalence of cardiovascular histopathology changes was 11.1% of compounds, with 53% recording a functional blood pressure or heart rate change. Correlations were assessed using the Mantel-Haenszel Chi-square trend test, identifying statistically significant associations between cardiac muscle pathology and (1) decreased blood pressure, (2) increased heart rate, and between cardiovascular vessel pathology and increased heart rate. Negative predictive values were high, suggesting few compounds cause repeat-dose cardiovascular structural change in the absence of functional effects in single-dose safety pharmacology studies. Therefore, observed functional changes could prompt moving (sub)chronic toxicology studies forward, to identify cardiovascular liabilities earlier in development, and reduce late-stage attrition.

Stimulation of the pelvic nerve increases bladder capacity in the PGE2 cat model of overactive bladder.

Selective electrical stimulation of the pudendal nerve exhibits promise as a potential therapy for treating overactive bladder (OAB) across species (rats, cats, and humans). More recently, pelvic nerve (PelN) stimulation was demonstrated to improve cystometric bladder capacity in a PGE2 rat model of OAB. However, PelN stimulation in humans or in an animal model that is more closely related to humans has not been explored. Therefore, our objective was to quantify the effects of PGE2 and PelN stimulation in the cat. Acute cystometry experiments were conducted in 14 α-chloralose-anesthetized adult, neurologically intact female cats. Intravesical PGE2 decreased bladder capacity, residual volume, threshold contraction pressure, and mean contraction pressure. PelN stimulation reversed the PGE2-induced decrease in bladder capacity and increased evoked external urethral sphincter electromyographic activity without influencing voiding efficiency. The increases in bladder capacity generated by PelN stimulation were similar in the rat and cat, but the stimulation parameters to achieve this effect differed (threshold amplitude at 10 Hz in the rat vs. twice threshold amplitude at 1 Hz in the cat). These results highlight the potential of PGE2 as a model of OAB and provide further evidence that PelN stimulation is a promising approach for the treatment of OAB symptoms.

Modulation of Calcitonin, Parathyroid Hormone, and Thyroid Hormone Secretion by Electrical Stimulation of Sympathetic and Parasympathetic Nerves in Anesthetized Rats.

The thyroid and parathyroid glands are dually innervated by sympathetic (cervical sympathetic trunk [CST]) and parasympathetic (superior laryngeal nerve [SLN]) nerve fibers. We examined the effects of electrical stimulation of efferent or afferent nerve fibers innervating the thyroid and parathyroid glands on the secretion of immunoreactive calcitonin (iCT), parathyroid hormone (iPTH), 3,3',5-triiodothyronine (iT3), and thyroxine (iT4) from the thyroid and parathyroid glands. In anesthetized and artificially ventilated rats, thyroid venous blood was collected. The rate of hormone secretion from the glands was calculated from plasma hormone levels, measured by ELISA, and the flow rate of thyroid venous plasma. SLNs or CSTs were stimulated bilaterally with rectangular pulses with a 0.5-ms width. To define the role of unmyelinated nerve fibers (typically efferent), the cut peripheral segments were stimulated at various frequencies (up to 40 Hz) with a supramaximal intensity to excite all nerve fibers. The secretion of iCT, iT3, and iT4 increased during SLN stimulation and decreased during CST stimulation. iPTH secretion increased during CST stimulation, but was not affected by SLN stimulation. To examine the effects of selective stimulation of myelinated nerve fibers (typically afferent) in the SLN, intact SLNs were stimulated with a subthreshold intensity for unmyelinated nerve fibers. iCT, iT3, and iT4 secretion increased during stimulation of intact SLNs at 40 Hz. These results suggest that excitation of myelinated afferents induced by low intensity and high frequency stimulation of intact SLNs promotes secretion of CT and thyroid hormones from the thyroid gland, potentially via reflex activation of parasympathetic efferent nerve fibers in the SLN.

Development and characterization of glutamyl-protected N-hydroxyguanidines as reno-active nitric oxide donor drugs with therapeutic potential in acute renal failure.

Acute renal failure (ARF) has high mortality and no effective treatment. Nitric oxide (NO) delivery represents a credible means of preventing the damaging effects of vasoconstriction, central to ARF, but design of drugs with the necessary renoselectivity is challenging. Here, we developed N-hydroxyguanidine NO donor drugs that were protected against spontaneous NO release by linkage to glutamyl adducts that could be cleaved by γ-glutamyl transpeptidase (γ-GT), found predominantly in renal tissue. Parent NO donor drug activity was optimized in advance of glutamyl adduct prodrug design. A lead compound that was a suitable substrate for γ-GT-mediated deprotection was identified. Metabolism of this prodrug to the active parent compound was confirmed in rat kidney homogenates, and the prodrug was shown to be an active vasodilator in rat isolated perfused kidneys (EC50 ~50 µM). The data confirm that glutamate protection of N-hydroxyguanidines is an approach that might hold promise in ARF.

Tetrahydrobiopterin analogues with NO-dependent pulmonary vasodilator properties.

Reduced NO levels due to the deficiency of tetrahydrobiopterin (BH(4)) contribute to impaired vasodilation in pulmonary hypertension. Due to the chemically unstable nature of BH(4), it was hypothesised that oxidatively stable analogues of BH(4) would be able to support NO synthesis to improve endothelial dysfunction in pulmonary hypertension. Two analogues of BH(4), namely 6-hydroxymethyl pterin (HMP) and 6-acetyl-7,7-dimethyl-7,8-dihydropterin (ADDP), were evaluated for vasodilator activity on precontracted rat pulmonary artery rings. ADDP was administered to pulmonary hypertensive rats, followed by measurement of pulmonary vascular resistance in perfused lungs and eNOS expression by immunohistochemistry. ADDP and HMP caused significant relaxation in vitro in rat pulmonary arteries depleted of BH(4) with a maximum relaxation at 0.3µM (both P<0.05). Vasodilator activity of ADDP and HMP was completely abolished following preincubation with the NO synthase inhibitor, L-NAME. ADDP and HMP did not alter relaxation induced by carbachol or spermine NONOate. BH(4) itself did not produce relaxation. In rats receiving ADDP 14.1mg/kg/day, pulmonary vasodilation induced by calcium ionophore A23187 was augmented and eNOS immunoreactivity was increased. In conclusion, ADDP and HMP are two analogues of BH(4), which can act as oxidatively stable alternatives to BH(4) in causing NO-mediated vasorelaxation. Chronic treatment with ADDP resulted in improvement of NO-mediated pulmonary artery dilation and enhanced expression of eNOS in the pulmonary vascular endothelium. Chemically stable analogues of BH(4) may be able to limit endothelial dysfunction in the pulmonary vasculature.

Endothelial dysfunction: from molecular mechanisms to measurement, clinical implications, and therapeutic opportunities.

Endothelial dysfunction has been implicated as a key factor in the development of a wide range of cardiovascular diseases, but its definition and mechanisms vary greatly between different disease processes. This review combines evidence from cell-culture experiments, in vitro and in vivo animal models, and clinical studies to identify the variety of mechanisms involved in endothelial dysfunction in its broadest sense. Several prominent disease states, including hypertension, heart failure, and atherosclerosis, are used to illustrate the different manifestations of endothelial dysfunction and to establish its clinical implications in the context of the range of mechanisms involved in its development. The size of the literature relating to this subject precludes a comprehensive survey; this review aims to cover the key elements of endothelial dysfunction in cardiovascular disease and to highlight the importance of the process across many different conditions.