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Biomedical subjects

J B Tuttle

Publications and source records attributed to J B Tuttle.

At least 37 records · Page 2Linked to original sources
PubMed1998

Mitochondria in sporadic amyotrophic lateral sclerosis.

Mitochondria are abnormal in persons with amyotrophic lateral sclerosis (ALS) for unknown reasons. We explored whether aberration of mitochondrial DNA (mtDNA) could play a role in this by transferring mitochondrial DNA (mtDNA) from ALS subjects to mtDNA-depleted human neuroblastoma cells. Resulting ALS cytoplasmic hybrids (cybrids) exhibited abnormal electron transport chain functioning, increases in free radical scavenging enzyme activities, perturbed calcium homeostasis, and altered mitochondrial ultrastructure. Recapitulation of defects previously observed in ALS subjects and ALS transgenic mice by expression of ALS mtDNA support a pathophysiologic role for mtDNA mutation in some persons with this disease.

1-Methyl-4-phenylpyridinium↗
PubMed1998

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.

Animals↗
PubMed1998

Mechanical stretch increases secretion of parathyroid hormone-related protein by cultured bladder smooth muscle cells.

Parathyroid hormone-related protein (PTHrP) immunoreactivity has been detected in the bladder and increases in response to dilatation secondary to obstruction. The hypothesis that PTHrP could be increased solely by stretch rather than other possible in vivo variables was tested by stretching cultured bladder smooth muscle cells and analyzing the culture medium for this protein. In response to mechanical stretch, PTHrP was increased in smooth muscle cell cultures. Immunoradiometric assay revealed maximal rates of secretion for the first eight hours. Comparison of percent change in PTHrP secretion of flexed cells for the various flex parameters revealed a difference (p = .006) when the degree of stretch (i.e. percent elongation) was altered. The protein synthesis inhibitor cycloheximide inhibited basal and stretch-induced PTHrP secretion. PTHrP (1-100 nM) relaxed carbachol-contracted bladder body and base by 15% and 45% respectively. PTHrP did not affect bladder contractions induced by potassium (124 mM) or alpha-beta MeATP (10 microM). Increased PTHrP secretion in response to stretch of smooth muscle raises the possibility of an autocrine action to relax the bladder during filling. PTHrP may also exert a paracrine action on vessels regulating blood flow during bladder filling or it may modulate neural activity.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

Spinal and peripheral mechanisms contributing to hyperactive voiding in spontaneously hypertensive rats.

The influence of noradrenergic mechanisms involved in micturition in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats was investigated using continuous cystometry in in vivo and in vitro studies on isolated bladder and urethral tissues. Compared with WKY rats, SHR had a significantly lower bladder capacity (SHR: 0.7 +/- 0. 05 ml; WKY rats: 1.3 +/- 0.06 ml; P < 0.001), micturition volume (SHR: 0.4 +/- 0.04 ml, WKY rats: 1.2 +/- 0.05 ml; P < 0.001), and an increased amplitude of nonvoiding (unstable) bladder contractions. The effects of intrathecal and intra-arterial doxazosin on cystometric parameters were more pronounced in SHR than in WKY rats. There was a marked reduction in nonvoiding contractions after intrathecal (but not intra-arterial) doxazosin in SHR. Norepinephrine (0.1 microM-1 mM) failed to evoke contractions in bladder strips from WKY rats, in contrast to a weak contractile response in SHR. The response to electrical field stimulation was significantly less in bladder strips from SHR than from WKY rats. In WKY rats, norepinephrine produced concentration-dependent inhibition (87 +/- 5%, n = 6) of nerve-evoked bladder contractions. Almost no inhibition (11 +/- 8%, n = 6) was found in SHR. Alterations in bladder function of SHR appear to be associated with changes in the noradrenergic control of the micturition reflex, in addition to an increased smooth muscle and decreased neuronal responsiveness to norepinephrine. The marked reduction in nonvoiding contractions after intrathecal doxazosin suggests that the bladder hyperactivity in SHR has at least part of its origin in supraspinal and/or spinal structures.

Animals↗
PubMed1997

Calcium homeostasis and reactive oxygen species production in cells transformed by mitochondria from individuals with sporadic Alzheimer's disease.

Alzheimer's disease (AD) is associated with defects in mitochondrial function. Mitochondrial-based disturbances in calcium homeostasis, reactive oxygen species (ROS) generation, and amyloid metabolism have been implicated in the pathophysiology of sporadic AD. The cellular consequences of mitochondrial dysfunction, however, are not known. To examine these consequences, mitochondrially transformed cells (cybrids) were created from AD patients or disease-free controls. Mitochondria from platelets were fused to rho0 cells created by depleting the human neuroblastoma line SH-SY5Y of its mitochondrial DNA (mtDNA). AD cybrids demonstrated a 52% decrease in electron transport chain (ETC) complex IV activity but no difference in complex I activity compared with control cybrids or SH-SY5Y cells. This mitochondrial dysfunction suggests a transferable mtDNA defect associated with AD. ROS generation was elevated in the AD cybrids. AD cybrids also displayed an increased basal cytosolic calcium concentration and enhanced sensitivity to inositol-1,4, 5-triphosphate (InsP3)-mediated release. Furthermore, they recovered more slowly from an elevation in cytosolic calcium induced by the InsP3 agonist carbachol. Mitochondrial calcium buffering plays a major role after this type of perturbation. beta-amyloid (25-35) peptide delayed the initiation of calcium recovery to a carbachol challenge and slowed the recovery rate. Nerve growth factor reduced the carbachol-induced maximum and moderated the recovery kinetics. Succinate increased ETC activity and partially restored the AD cybrid recovery rate. These subtle alterations in calcium homeostasis and ROS generation might lead to increased susceptibility to cell death under circumstances not ordinarily toxic.

Adenine Nucleotides↗
PubMed1997

MPP+ induced apoptotic cell death in SH-SY5Y neuroblastoma cells: an electron microscope study.

PD is a common, late-onset neurodegenerative disorder that results in part from the gradual loss of dopaminergic neurons in the substantia nigra pars compacta. The neurotoxin MPTP can induce PD-like clinical symptomatology and neuropathological destruction and, thus, has been used as a PD model. The human neuroblastoma cell line SH-SY5Y possesses many of the qualities of human neurons and, as such, has served as a model for them. Apoptosis is the mode of cell death induced in SH-SY5Y cells by MPTP, and this was confirmed with nick end labeling and bisbenzimide staining. Transmission electron microscopic analysis of the ultrastructural changes occurring in neurotoxin exposed SH-SY5Ys revealed many morphological characteristics consistent with apoptosis. These changes included plasmalemmal blebbing, altered cytosolic density, nuclear condensation and fragmentation, pronounced vacuole formation, ribosomal dispersion, and the disappearance of the golgi complex, microtubules, and smooth endoplasmic reticulum. Limited amounts of rough endoplasmic reticulum and mitochondria exhibited normal morphology throughout the apoptotic changes but then were disrupted during secondary necrotic changes. The in vitro induction of apoptosis by a parkinsonism neurotoxin might be reflective of the mechanisms of in vivo nigral degeneration occurring during PD.

1-Methyl-4-phenylpyridinium↗
PubMed1997

Synergistic increase in nerve growth factor secretion by cultured vascular smooth muscle cells treated with injury-related growth factors.

Vascular smooth muscle (VSM) cells comprise one of the primary targets of the sympathetic nervous system and have been shown to secrete nerve growth factor (NGF). There is increasing evidence that changes in the levels of NGF in the adult may underlie certain pathological conditions. To investigate the potential role of altered NGF production in vascular disease, VSM cell cultures were treated with injury-related growth factors and the culture medium was assayed for NGF using a two-site enzyme-linked immunosorbent assay (ELISA). Platelet-derived growth factor (PDGF), a potent VSM mitogen, caused a dose-dependent increase in NGF secretion. After 4 hr, PDGF-treated cultures contained 10 times more NGF than control cultures. NGF release remained elevated for 48 hr, but the peak secretion occurred in the first 12 hr after treatment. Transforming growth factor beta (TGF-beta) caused a fivefold increase in NGF at 4 hr when added alone, but synergized with PDGF yielding approximately 50 times more NGF than control cultures. TGF-beta and epidermal growth factor (EGF) also displayed synergism. In contrast, basic fibroblast growth factor (bFGF), which had a modest effect alone, appeared to be additive with TGF-beta. Similarly, interleukin 1-beta (IL-1 beta), which mediates increased NGF synthesis in sciatic nerve lesions (Lindholm et al.: Nature 330:658-659, 1987), showed no synergism with TGF-beta.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

Thrombin regulates nerve growth factor secretion from vascular, but not bladder smooth muscle cells.

The production of nerve growth factor (NGF) in peripheral organs may play a role in the pathophysiology of hypertension and in obstructive disorders of the bladder outlet. We have been examining the cellular processes of NGF delivery and secretion in smooth muscle. NGF secretion from vascular smooth muscle cells (VSMCs) cultured from genetically hypertensive (WKHT), hyperactive (WKHA), and a control Wistar rat strain were assayed using a two-site ELISA of the culture media. Bladder smooth muscle cells (BSMCs) from the Wistar strain were also studied. The serine protease, thrombin, increased NGF secretion from all types of VSMCs but had no effect on Wistar BSMCs. The thrombin-mediated increase in NGF secretion was prevented by actinomycin D and cycloheximide, suggesting that RNA transcription and protein synthesis are required. The effect of thrombin was additive with a phorbol ester-induced elevation in NGF secretion rates from 4 to 6 h and was attenuated by a 24-h downregulation of protein kinase C. These results suggest that extracellular protease activity may regulate NGF secretion in smooth muscle. Thrombin may act in response to vascular injury, increasing NGF secretion from VSMCs, initiating VSMC migration, and preparing the VSMCs for reinnervation following an insult.

Animals↗
PubMed1997

Altered calcium homeostasis in cells transformed by mitochondria from individuals with Parkinson's disease.

Parkinson's disease may be linked to defects in mitochondrial function. Mitochondrially transformed cells (cybrids) were created from Parkinson's disease patients or disease-free controls. Parkinson's disease cybrids had 26% less complex I activity, but maintained comparable basal calcium and energy levels. Parkinson's disease cybrids recovered from a carbachol-induced increase in cytosolic calcium 53% more slowly than controls even with lanthanum and thapsigargin blockade. Inhibition of complex I with the Parkinson's disease-inducing metabolite 1-methyl-4-phenylpyridinium (MPP+) similarly reduced the rate of recovery after carbachol. This MPP(+)-induced reduction in recovery rates was much more pronounced in control cybrids than in Parkinson's disease cybrids. Parkinson's disease cybrids had less carbonyl cyanide m-chlorophenylhydrazone-releasable calcium. Bypassing complex I with succinate partially restored Parkinson's disease cybrid, and MPP+ suppressed control cybrid recovery rates. The subtle alteration in calcium homeostasis of Parkinson's disease cybrids may reflect an increased susceptibility to cell death under circumstances not ordinarily toxic.

1-Methyl-4-phenylpyridinium↗
PubMed1997

Regulation of nerve growth factor secretion in smooth muscle cells cultured from rat bladder body, base and urethra.

PURPOSE: Interest in the regulation of nerve growth factor (NGF) production in the urinary tract derives from its probable involvement in obstructive, inflammatory and developmental disorders. This study examines receptor-mediated stimuli that alter NGF production in cells of the lower urinary tract. MATERIALS AND METHODS: Cells were isolated and cultured from the bladder body, base and urethra, confirmed as smooth muscle type by alpha-actin expression, and examined for growth rate and NGF secretion in response to autonomic agonists, cytokines, neuropeptides and growth factors. NGF secreted into the culture medium was quantitated via 2-site enzyme-linked immunoassay. Regional tissue contents of NGF and norepinephrine (NE) were also measured. Only statistically significant differences (Student's t test, p <0.05) are reported. RESULTS: Cultured urinary tract cells derived from different regions varied in growth rate and NGF secretory activity. Bladder body secreted less NGF than base, and base less than urethra. A similar gradient in growth rate occurred in vitro, with urethral cells most active. However, no regional differences were found in bladder tissue NGF content despite significant variations in NE levels. Platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) were among the most potent stimuli to NGF production by cultured cells while cAMP linked receptors and eicosinoids inhibited NGF output. CONCLUSIONS: A complex system of regionally specific and stimulus-specific control regulates the production of NGF by urinary tract cells. While tissue levels of NGF do not correlate with the density of noradrenergic innervation, bladder innervation is sufficiently dynamic to respond to changes in NGF production and to participate in pathophysiology.

Animals↗
PubMed1996

Origin and functional consequences of the complex I defect in Parkinson's disease.

The mitochondrial electron transport enzyme NADH:ubiquinone oxidoreductase (complex I), which is encoded by both mitochondrial DNA and nuclear DNA, is defective in multiple tissues in persons with Parkinson's disease (PD). The origin of this lesion and its role in the neurodegeneration of PD are unknown. To address these questions, we created an in vitro system in which the potential contributions of environmental toxins, complex I nuclear DNA mutations, and mitochondrial DNA mutations could be systematically analyzed. A clonal line of human neuroblastoma cells containing no mitochondrial DNA was repopulated with mitochondria derived from the platelets of PD or control subjects. After 5 to 6 weeks in culture, these cytoplasmic hybrid (cybrid) cell lines were assayed for electron transport chain activities, production of reactive oxygen species, and sensitivity to induction of apoptotic cell death by 1-methyl-4-phenyl pyridinium (MPP+). In PD cybrids we found a stable 20% decrement in complex I activity, increased oxygen radical production, and increased susceptibility to 1-methyl-4-phenyl pyridinium-induced programmed cell death. The complex I defect in PD appears to be genetic, arising from mitochondrial DNA, and may play an important role in the neurodegeneration of PD by fostering reactive oxygen species production and conferring increased neuronal susceptibility to mitochondrial toxins.

1-Methyl-4-phenylpyridinium↗
PubMed1996

Immunity to nerve growth factor prevents afferent plasticity following urinary bladder hypertrophy.

PURPOSE: The goal of this investigation was to examine the effect of immunity to nerve growth factor (NGF) on alterations in sensory nerves from the urinary bladder in the dorsal root ganglia (DRG) and their projections to the L6/S1 spinal cord following urethral obstruction in the rat. MATERIALS AND METHODS: Female Wistar rats were immunized to murine 2.5S NGF, then obstructed by partial urethral ligation for 6 weeks. Retrograde axonal tracing with FluoroGold and WGA-HRP was used to measure areas of bladder DRG cells and afferent projections in the sacral spinal cord. Multiunit activity on bladder nerves allowed recording of micturition reflexes. Immunohistochemical staining for growth associated protein (GAP)-43 in the sacral parasympathetic nucleus (SPN) was used to assess potential growth or activity of axons in the spinal cord. Voiding frequencies were then measured in awake obstructed and NGF immune-obstructed rats. RESULTS: Immunity to NGF prevented obstruction-induced hypertrophy of DRG neurons, reduced retrograde axonal labeling of sacral afferent projections, eliminated enhancement of a spinal micturition reflex and abolished the increased GAP-43 expression in the SPN. Immunity to NGF prevented the urinary frequency that accompanies obstruction. CONCLUSIONS: Our results demonstrate that obstruction of the bladder elicits structural and functional plasticity in afferents as a result of ongoing neurotrophic interactions. Neurotrophic interactions offer a potential mechanism whereby symptoms and bladder hyperactivity develop with obstruction associated with benign prostatic hyperplasia.

Animals↗
PubMed1996

Characterization of laterality of innervation of the rat bladder.

Retrograde axonal tracers were used to assess the laterality of bladder innervation which is unknown. Fluoro-Gold and Fast Blue were injected into the left and right bladder walls, respectively of male Wistar rats. The major pelvic (MPG), pelvic accessory (PAG), and dorsal root (DRG) ganglia were then removed and analyzed. 59% and 33% of the total labelled MPG cells were found in the ipsilateral and contralateral MPG (efferents), respectively. 65% and 31% of the total labelled PAG cells were found in the ipsilateral and contralateral PAG (efferents) respectively. The L1/L2 and L6/S1 DRG each received similar amount of ipsilateral innervation (45-52%) based on total labelled cells at each DRG level. 8%, 4%, and 4% of labelled cells in the MPG, PAG and DRG, respectively, contained both Fluoro-Gold and Fast Blue consistent with bilateral input originating from a single neuron. Immunohistochemistry showed that 40% of retrogradely labelled PAG cells were positive for tyrosine hydroxylase, far exceeding what has been shown for the MPG. Thus, assuming limited diffusion of tracers, the bladder receives a substantial crossed innervation especially in bladder afferents. This redundancy and bilateral neural input may contribute to preservation of bladder function and sensation even after extensive pelvic surgery.

Amidines↗
PubMed1995

Altered signalling in vascular smooth muscle from spontaneously hypertensive rats may link medial hypertrophy, vessel hyperinnervation and elevated nerve growth factor.

1. Secretion of nerve growth factor (NGF) by cultured vascular smooth muscle cells (VSMC) derived from spontaneously hypertensive rats (SHR) and the normotensive Wistar-Kyoto (WKY) strain was measured via two site immunoassay (ELISA). 2. Basal NGF secretion rates of quiescent SHR VSMC in serum-free culture medium were elevated compared to similar WKY VSMC. 3. SHR VSMC displayed increased NGF secretion in response to activation of sympathetic neurotransmitter receptors while VSMC of WKY were largely unresponsive to the agents (phenylephrine, isoproterenol, alpha-beta-methyl ATP, neuropeptide Y). 4. Mitogenic stimulation with platelet-derived growth factor (PDGF) raised SHR NGF secretion rates almost three times more than PDGF increased WKY secretion. 5. SHR VSMC also failed to demonstrate normal inhibitory control over NGF secretion seen in WKY and previously in Sprague-Dawley and Wistar strain VSMC with adenylate cyclase activation and down-regulation of protein kinase C. High concentrations of forskolin stimulated, instead of inhibiting, secretion in SHR. Stimulation was also seen after pretreatment with phorbol ester for 24 h while this inhibited secretion in the WKY. 6. These results confirm that the SHR VSMC are hyperresponsive to growth stimuli such as contractile agonists and mitogens. This hyperresponsiveness includes an abnormal control over NGF secretion such that normally inhibitory treatments stimulate NGF output in the SHR. 7. Because the SHR demonstrates important defects in the major intracellular growth-signalling systems that also regulate NGF output and vessel innervation, the predicted result of the defects is a destructive feed-forward cycle of growth and innervation. This is the SHR phenotype in vivo.

Animals↗
PubMed1995

Nerve growth factor, vessel innervation and hypertensive progression in the inbred Dahl SS/Jr and SR/Jr rats.

1. To test whether the inbred Dahl salt-sensitive hypertensive rat strain shares disturbed vessel innervation with the spontaneously hypertensive rat (SHR) model, highly innervated and sparsely innervated tissues from the SR/Jr and SS/Jr strains at several ages were assayed for tissue norepinephrine (NE) content and nerve growth factor (NGF). 2. Only two significant differences were found: (i) 1 week old SS/Jr rat kidneys had more NGF than SR/Jr kidneys; and (ii) six week old mesenteric arteries from SS/Jr contained significantly more NE than those in the SR/Jr animals. 3. The differences are in the expected direction for vessel hyperinnervation as a consequence of high vessel NGF, but the data do not support the hypothesis of a central role for vessel NGF and innervation in the hypertensive progression of the Dahl strain. The vessels of young Dahl inbred rat strains do not display the same degree of vessel hyperinnervation associated with hypertension as found in the young SHR compared to WKY strains. 4. The Dahl strain may lack the disturbed NGF metabolism of the SHR. This might suggest that the Dahl inbred strains and the SHR represent two distinct genetic mechanisms that predispose to hypertension via essentially independent processes and that all forms will evidence at least mildly altered vessel innervation.

Animals↗