Stretch-activated signaling of nerve growth factor secretion in bladder and vascular smooth muscle cells from hypertensive and hyperactive rats.

Elevated vascular (VSMC) and bladder smooth muscle (BSMC) NGF are associated with altered visceral innervation in the spontaneously hypertensive rat (SHR: hypertensive, behaviorally hyperactive) compared with control Wistar-Kyotos (WKYs). Stretch stimulates increased NGF production in BSMCs. To elucidate whether stretch induces NGF synthesis in VSMCs, and to determine if disturbances in stretch-mediated NGF production contribute to the elevated tissue levels of NGF in SHRs, we subjected VSMCs and BSMCs cultured from four established inbred rat strains (WKY, WKHA: hyperactive; SHR and WKHT: hypertensive) to several stretch paradigms. For VSMCs, acute and cyclic stretch affected cells derived from hypertensive rats (80-100% increase over control) but not from normotensive strains. For BSMCs, cyclic and static stretch increased NGF secretion in all four strains, but had a two- to threefold greater effect in cells from SHRs and WKHTs (increase up to 600%) at early time points. At later time points of a 24-h experimental period, stretch increased NGF output up to 400% in SHR and WKHA cultures. Thus, defects that influence early induction of stretch-mediated SHR NGF secretion cosegregate with the hypertensive phenotype. Stretch-gated ion channel inhibitors, voltage-gated ion channel inhibitors, and protease inhibitors failed to affect stretch-induced BSMC NGF secretion. In contrast, gene transcription, intracellular calcium, protein kinase C (PKC), and autocrine release of an unknown factor may play a role in the elevated NGF secretion observed in smooth muscle from hypertensive animals. Altered stretch-induced smooth muscle NGF secretion may contribute to the augmented vascular and bladder NGF content associated with high blood pressure and hyperactive voiding in SHRs.

Calcium homeostasis and nerve growth factor secretion from vascular and bladder smooth muscle cells.

Bladder and vascular smooth muscle cells cultured from four rat strains (WKY, SHR, WKHA, WKHT) differing in rates of nerve growth factor (NGF) production were used to determine whether a relationship exists between intracellular calcium and NGF secretion. Basal cytosolic calcium was related to basal NGF secretion rates in bladder and vascular smooth muscle cells from all four strains with the exception of WKHT bladder muscle cells. Thrombin is a calcium-mobilizing agent and increases NGF production from vascular but not bladder smooth muscle cells. Strain differences were found in the magnitude of the calcium peak induced by thrombin in vascular smooth muscle cells, but these differences did not correlate with NGF secretion. Thrombin caused a calcium response in bladder smooth muscle cells without influencing NGF production. Quenching the calcium transient with a calcium chelator had no effect on thrombin-inducted NGF secretion rates in vascular smooth muscle cells. Thus, basal intracellular calcium may establish a set point for NGF secretion from smooth muscle. In addition, transient elevations in cytosolic calcium were unrelated to the induction of NGF output.

The spontaneously hypertensive rat: insight into the pathogenesis of irritative symptoms in benign prostatic hyperplasia and young anxious males.

Recent epidemiological studies have shown that hypertensive men are more likely to undergo surgical intervention for irritative voiding symptoms from BPH than age-matched controls. Indeed, noradrenergic nerves which regulate vascular tone also participate in the functional component of bladder outlet obstruction due to BPH. Newer, less invasive therapies for BPH such as thermal therapy can relieve symptoms yet do not eliminate obstruction based on urodynamic studies. Coincidentally, drugs such as alpha-adrenoceptor antagonists, which have been thought to relieve obstruction due to a peripheral effect, can be given intrathecally in animals to relieve urinary frequency due to obstruction. Taken together these observations implicate both peripheral and central sympathetic pathways in the motor control of the urinary bladder especially with disease states. We have used the hypertensive and behaviourally hyperactive spontaneously hypertensive rat (SHR), to investigate the roles sympathetic pathways or micturition. Elevated nerve growth factor (NGF) derived from vascular and bladder smooth muscle cells of the SHR appears to direct morphological, biochemical, and functional changes. The increase in NGF can apparently be explained by stabilization of its mRNA leading to increased synthesis in NGF. Bladders from SHRs develop a profuse noradrenergic hyperinnervation compared with the control WKY strain. Since afferents supplying the SHR bladder are hypertrophied, changes in afferent pathways are also likely. These differences in innervation and NGF in the SHR may explain changes in function. SHRs void 3 times as frequently as their genetic controls. Urinary frequency can be reduced by alpha-adrenoceptor antagonists. Cystometrograms performed in SHRs reveal lower bladder capacities and micturition volumes and the presence of unstable contractions compared with the WKY rat. Intrathecal, rather than intra-arterial administration of the alpha-adrenoceptor antagonist doxazosin reduces unstable contractions in the SHR. In vitro muscle bath studies have shown enhanced responses of SHR bladder smooth muscle to alpha-adrenoceptor agonists. It is likely that upregulation of NGF production causes sensory and possibly noradrenergic pathways to elicit hyperactive voiding. Increase in NGF in the adult bladder due to pathological conditions yields similar, yet distinct, consequences for voiding behaviour and innervation. Likewise, increased NGF in adult bladders following obstruction or inflammation triggers neuronal hypertrophy, enhanced reflex activity and urinary frequency. In contrast to the SHR, hyper-innervation is not observed. Moreover, peripheral or spinal alpha-adrenoceptor blockade eliminates urinary frequency following obstruction. These observations support the role for sympathetic pathways in the motor function of the bladder, especially in congenital or adult disease states. A similar process may underlie the neuroplasticity involved in alterations after obstruction or inflammation of the lower urinary tract in humans. The SHR strain raises the possibility that a common genetic defect exists capable of predisposing to both hypertension and overactivity of the urinary bladder. Whether a genetic predisposition to sustained bladder overactivity in response to inflammatory stimuli in obstruction exists in humans is an intriguing prospect.

Altered NGF regulation may link a genetic predisposition for hypertension with hyperactive voiding.

PURPOSE: Hyperactive voiding and elevated smooth muscle NGF output are traits of the spontaneously hypertensive rat (SHR). Elevated target-derived NGF is associated with hypertension and hyperactive voiding in SHRs. In the present study, we tested for possible genetic links between hypertension, hyperactive voiding and augmented bladder smooth muscle cell (BSMC) NGF secretion. MATERIALS AND METHODS: We crossed SHRs with WKYs to produce a gene segregating F2 population. We measured F2 mean arterial blood pressure (BP) and six-hour voiding frequency. BSMCs were cultured from 'Low BP F2s' (95+/-2) and 'High BP F2s' (141+/-3 mm. Hg) and conditioned medium tested for NGF with a two-site ELISA. The NGF regulators isoproterenol, platelet-derived growth factor (PDGF) and phorbol-12-myristate-13-acetate were tested in F2 BSMC cultures. RESULTS: A positive correlation (r = 0.75) between blood pressure and voiding frequency existed in this F2 population. As BP rose voiding frequency increased and volume per void decreased such that there were no significant changes in total urine voided (Low BP F2s: 1.0+/-0.5; High BP F2s: 6.2+/-0.5 voids/6 hours). Low BP F2s (2.0+/-0.2) secreted NGF at a higher basal rate than High BP F2s (0.7+/-0.1 fg NGF/hr/100 cells). However, High BP F2s (1,620 and 3,850) were oversensitive to isoproterenol and PDGF-induced increases in NGF output, compared with Low BP F2s (219 and 1,282% control, respectively). CONCLUSIONS: Elevated tissue NGF due to a hypersensitivity to NGF regulating stimuli, rather than alterations in basal NGF, may genetically link hypertension and hyperactive voiding.

Effects of growth rate and cell density on nerve growth factor secretion in cultures of vascular and bladder smooth muscle cells from hypertensive and hyperactive rats.

Elevated target-derived smooth muscle nerve growth factor (NGF) and resultant neurogenic plasticity are associated with both hypertension and hyperactive voiding in spontaneously hypertensive rats (SHRs: hypertensive, behaviorally hyperactive). In culture, vascular (VSMCs) and bladder (BSMCs) smooth muscle cells derived from SHRs secrete higher levels of NGF, proliferate more rapidly, and achieve higher density at confluence than do control Wistar-Kyoto (WKY) cells. To elucidate growth-related contributions to the elevated tissue NGF observed in SHRs, we examined vascular VSMC and BSMC NGF secretion in two inbred cell lines (WKHTs, hypertensive; WKHAs, hyperactive) derived from SHRs and WKYs to assess the phenotypic association of altered NGF metabolism with either hypertension or behavioral hyperactivity. Cell density, rather than growth rates, was the most important factor with respect to NGF secretion. VSMC density varied such that WKHT=SHR>WKY= WKHA, higher VSMC density being associated with higher NGF output. However, in BSMC cultures, NGF output was the lowest in high density cell lines, with WKHT>SHR>WKY>WKHA. SHR BSMCs had the second highest cell density and NGF secretion level. Elevated packing density, presumably because of a lack of contact inhibition, co-segregated with the hypertensive phenotype in both VSMCs and BSMCs. Thus, dysfunctional smooth muscle growth characteristics may contribute to the augmented vascular and bladder NGF content associated with high blood pressure and hyperactive voiding in SHRs.

Arterial nerve growth factor (NGF) mRNA, protein, and vascular smooth muscle cell NGF secretion in hypertensive and hyperactive rats.

Elevated levels of nerve growth factor (NGF) protein and NGF mRNA have been reported in the vessels of spontaneously hypertensive rats (SHR: hypertensive, hyperactive) compared to Wistar-Kyoto (WKY) rats. Elevated NGF may be involved in the development of hypertension in SHRs. We examined vascular NGF mRNA and protein content and the regulation of NGF secretion by vascular smooth muscle cells (VSMCs) from two inbred strains (WKHT: hypertensive; WKHA: hyperactive) derived from SHRs and WKYs. Our goal was to determine if receptor-mediated defects in NGF regulation play a role in increased secretion of VSMC NGF from hypertensive animals. Tissue NGF mRNA content was determined by competitive, quantitative RT-PCR. Tissue NGF and NGF content in cultured VSMC-conditioned medium was quantified using a two-site ELISA. Tail artery NGF mRNA was elevated in WKHTs compared to WKHAs. Tissue NGF protein was elevated in WKHT aorta, mesenteric, and tail artery compared to WKHAs. Pharmacologically induced increases in NGF output were blocked with inhibition of transcription or protein synthesis. Basal NGF secretion by WKHT VSMCs was significantly higher than WKHAs. The observed increases in VSMC NGF output in SHRs over WKYs in response to beta-adrenergic agents are not preserved in the WKHT:WKHA comparison. Protein kinase C-dependent increases in SHR VSMC NGF appear in both WKHTs and WKHAs. In contrast, elevated NGF levels due to disturbances in alpha-adrenergic, peptidergic, and purinergic control of NGF output are features common to both genetic models of hypertension (SHR and WKHT). These results suggest that the defect in smooth muscle NGF metabolism observed in SHRs cosegregates with a hypertensive rather than a hyperactive phenotype. Moreover, altered receptor-mediated regulation (alpha-adrenergic, peptidergic, and purinergic) of VSMC NGF production may contribute to elevated vascular tissue NGF, suggesting a mechanism leading to the high levels of NGF associated with hypertension in SHRs and WKHTs.

Altered regulation of bladder nerve growth factor and neurally mediated hyperactive voiding.

Elevated bladder smooth muscle cell (BSMC) nerve growth factor (NGF) secretion and related neuroplasticity are associated with hyperactive voiding in spontaneously hypertensive rats (SHRs: hypertensive, behaviorally hyperactive), compared with control Wistar-Kyotos (WKYs). We used two inbred strains (WKHT: hypertensive; WKHA: hyperactive) to further investigate this phenomenon. WKHA BSMCs secreted higher basal levels of NGF than WKHT BSMCs. Antagonists did inhibit NGF output in WKHA but not WKHT cultures. Thus augmented basal secretion of NGF cosegregates with a hyperactive phenotype, whereas a lack of regulatory inhibition of NGF output cosegregates with a hypertensive phenotype. Bladder norepinephrine content paralleled NGF content, with WKHTs > SHRs > WKHAs > WKYs, providing evidence that a lack of inhibition is the greatest contributor to elevated bladder NGF and noradrenergic innervation. Protein kinase C (PKC) agonists affected NGF production differentially depending on strain, suggesting that altered PKC signaling may contribute to strain differences in NGF secretion. Finally, 6-h voiding frequency differed between the strains, with SHRs > WKHTs = WKHAs > WKYs. Thus aspects of both the hypertensive and hyperactive phenotypes may be associated with elevated SHR bladder NGF and hyperactive voiding.

Neurally mediated hyperactive voiding in spontaneously hypertensive rats.

The development of hypertension in spontaneously hypertensive rats (SHR) and hyperactive voiding in rats with urethral obstruction are characterized by abnormal smooth muscle growth, increased tissue levels of nerve growth factor (NGF) and altered patterns of innervation. The present study was undertaken to determine if bladder smooth muscle from SHRs contains and secretes elevated levels of NGF, and if so, whether the augmented NGF contributes to changes in bladder innervation and function without tissue hypertrophy. Voiding behavior was monitored using specially designed metabolic cages. NGF levels in tissue homogenates and conditioned cell culture media were measured by ELISA. NGF mRNA in cultured bladder smooth muscle cells (BSMCs) was quantified using reverse transcriptase PCR. Noradrenergic innervation was assessed by staining with glyoxylic acid and assaying norepinephrine (NE) content in bladders with high performance liquid chromatography. SHRs voided more frequently than WKY rats. NGF content was higher in bladders from adult SHRs when compared to Wistar-Kyoto normotensive rats (WKYs). No significant difference in NGF mRNA content was observed between SHR and WKY BSMCs. However, SHR BSMCs secreted NGF at a higher rate and amount per unit mRNA than did WKY BSMCs. SHR bladders contained more NE and were more densely stained for catecholaminergic fibers than bladders from WKY rats. The results support the hypothesis that elevated NGF secretion by bladder smooth muscle is associated with hyperinnervation of bladder and hyperactive voiding in SHRs. Thus, the SHR strain may represent a genetic model to study changes in bladder function resulting from altered patterns of innervation.

Efferent and afferent neuronal hypertrophy associated with micturition pathways in spontaneously hypertensive rats.

Elevated nerve growth factor secreted by bladder smooth muscle may be associated with noradrenergic hyperinnervation of the bladder and hyperactive voiding in spontaneously hypertensive rats (SHR) and rats with bladder outlet obstruction. The present study was undertaken to determine if changes occur in efferent and afferent pathways supplying the SHR bladder similar to those in rats with bladder outlet obstruction. Fluoro-Gold (FG) retrograde tracing studies were conducted to examine the postganglionic efferent limb (major pelvic ganglion; MPG) and sensory afferent limb (L1, L2, L6, and S1 dorsal root ganglion; DRG) of the micturition reflex pathway of the SHR and Wistar-Kyoto (WKY) normotensive rat. A significant increase in cross sectional area profiles for labeled neurons in the MPG was observed in SHRs (830.5 +/- 9.0 microns2) as compared to WKYs (736.3 +/- 16.6 microns2). Neuronal cell areas in L2 (1,010.9 +/- 18.6 microns2) and S1 (1,024.6 +/- 28.3 microns2) of SHRs were significantly larger than those of WKYs (L2, 865.3 +/- 12.6 microns2, S1, 778.3 +/- 11.2 microns2). There was an increase in number of labeled cells in L6 within SHRs over WKYs. These results provide evidence that both efferent and afferent changes in neuronal innervation of the bladder occur in SHRs. The SHR strain may represent a genetic model to study changes in micturition reflex pathways that result from alterations in neuronal morphology such as those that occur with urethral outlet obstruction.

Development of 5'-nucleotidase staining in the olfactory bulbs of normal and naris-occluded rats.

The distribution of the adenosine-producing ecto-enzyme 5'-nucleotidase was investigated histochemically in the developing rat olfactory bulb. Rat pups underwent either unilateral surgical occlusion of the right external naris or sham surgery on postnatal day 1. At 10, 20, or 30 days postpartum, horizontal sections of the olfactory bulb were reacted histochemically to reveal the locus and intensity of 5'-nucleotidase activity. Relative staining levels were determined by optical densitometry in standardized bulb regions. A marked, age-related increase in staining density was observed. Reaction product was found primarily in neuropil areas. The P10 and P20 control animals did not exhibit right/left differences in bulb staining; however, some laterality was observed in P30 animals. Inter-glomerular and regional variations were observed throughout the developmental period, including (1) differences between neighboring glomeruli; (2) a gradient in the dorsal-ventral axis of the bulb; and (3) a higher staining density in the medial-caudal portion of the bulb. In subjects with occluded nares, asymmetries in right/left bulb 5'-nucleotidase staining patterns were detected throughout development. Bulbs ipsilateral to the blocked nares exhibited increased staining density, suggesting that the procedure enhanced enzymatic activity. Understanding these variations in 5'-nucleotidase staining may be important for a complete understanding of the mechanisms of olfactory bulb maturation and may give insight into the possible role of this enzyme in synaptic malleability during nervous system development and regeneration.