David B. Clemow

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 α‐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.

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.

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. J. Cell. Physiol. 183:289–300, 2000.

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.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.

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 μm2) as compared to WKYs (736.3 ± 16.6 μm2). Neuronal cell areas in L2 (1,010.9 ± 18.6 μm2) and S1 (1,024.6 ± 28.3 μm2) of SHRs were significantly larger than those of WKYs (L2, 865.3 ± 12.6 μm2; S1, 778.3 ± 11.2 μm2). 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. Neurourol. Urodynam. 16:293–303, 1997.

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

Abstract 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.

Observations from the Spontaneously Hypertensive Rat

An association between hypertension (HTN) and symptoms from benign prostatic hyperplasia (BPH) has been reported.1–4 Nearly a third of men undergoing treatment of prostatism are hypertensive. This increased prevalence of HTN exceeds that for age-matched cohorts. Such an association between these two seemingly independent disorders is intriguing because it raises the possibility of a common underlying mechanism- namely over-activity of the sympathetic nervous system and/or aging. In line with this reasoning, young anxious males can exhibit episodic rises in systemic blood pressure as well as the voiding symptoms urgency, frequency and slow urinary stream similar to men with prostatism. These anxiety-related symptoms have also been attributed to sympathetic nerves and adrenergic mechanisms.

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

Abstract. 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.

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.