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J C Hardwick

Publications and source records attributed to J C Hardwick.

At least 19 recordsLinked to original sources
PubMed2006

Altered bone morphogenetic protein signalling in the Helicobacter pylori-infected stomach.

Morphogens regulate epithelial cell fate decisions in the adult gastrointestinal tract. The authors hypothesized that influx of inflammatory cells into the lamina propria may disturb the normal expression gradients of morphogens (morphogenetic landscape) in gastrointestinal epithelia. Changes in the activity of the bone morphogenetic protein (BMP) pathway in normal and Helicobacter pylori-infected gastric mucosa were therefore examined. It is shown that BMP receptors, the activated (phosphorylated) form of the intracellular BMP signal transduction protein SMAD1, and BMP target ID2 all localize to gastric epithelial cells that are at the end of the axis of epithelial renewal in normal mucosa. Colonization of human gastric mucosa with H. pylori was associated with an increase in BMP2 expression due to influx of inflammatory cells that produce BMP2. Furthermore, whereas no BMP4 was detected in the normal antrum, focal infiltrates of BMP4-expressing cells were found in the H. pylori-infected stomach. This influx of BMP-expressing cells was associated with an increase in epithelial BMP signalling. Interestingly, a shift in activity of the BMP pathway was observed towards the precursor cell compartment (isthmus) of the gastric units. Thus, H. pylori infection results in an influx of inflammatory cells that disturb the normal activity gradient of a morphogenetic pathway with an established role in epithelial cell fate regulation. The data suggest that morphological changes in epithelial histology may result from alterations in the morphogenetic landscape secondary to changes in the cellular composition of the lamina propria.

Bone Morphogenetic Protein 2↗
PubMed2001

Regulation of parasympathetic neurons by mast cells and histamine in the guinea pig heart.

The potential interaction between the immune system and the autonomic nervous system was examined in the cardiac ganglia of guinea pigs. Intracellular voltage recordings were used to determine the effects of mast cell degranulation on the membrane properties of parasympathetic neurons in animals actively sensitized to ovalbumin. Stimulation of mast cell degranulation by perfusion with ovalbumin (10 micrograms/ml) produced a depolarization and increase in the excitability of intracardiac neurons. These effects could be mimicked by histamine application, either by perfusion (10 microM) or by local pressure application (100 microM, 1-2 s application). In either case, histamine application resulted in a similar membrane depolarization and increase in excitability. Immunohistochemical experiments demonstrated that histamine-immunoreactive mast cells are located in close proximity to parasympathetic postganglionic neurons. The histamine response was not due to release of other neurotransmitters from adjacent nerve terminals and both the depolarization and increase in excitability were inhibited by the H1 antagonist, pyrilamine (300 nM), and were unaffected by the H2 antagonist cimetidine (5 microM). Incubation of cardiac ganglion preparations from sensitized animals with pyrilamine prior to ovalbumin perfusion resulted in the inhibition of both the depolarization and increase in excitability. These results demonstrate that mast cell degranulation, and the subsequent release of histamine, results in the stimulation of intracardiac neurons via the activation of H1 receptors. Thus, local inflammatory reactions in the cardiac tissue can lead to the rapid activation of parasympathetic neurons, thereby altering cardiac function.

Action Potentials↗
PubMed2001

NF-kappaB, p38 MAPK and JNK are highly expressed and active in the stroma of human colonic adenomatous polyps.

The factors that govern the progression from colonic adenomatous polyp to colon cancer are poorly understood. The observation that NSAIDs act as chemopreventative agents and reduce the size of colonic polyps suggests the involvement of inflammatory signalling, but inflammatory signalling in colonic polyps has not been studied. We investigated the expression of the active forms of NF-kappaB, JNK and p38 MAPK using immunohistochemistry with activation specific antibodies in human colonic adenomas. We show that active NF-kappaB is seen in stromal macrophages that also express COX-2 and TNF-alpha, active JNK is seen in stromal and intraepithelial T-lymphocytes and periendothelial cells of new blood vessels, and active p38 MAPK is most highly expressed in macrophages and other stromal cells. These results demonstrate the presence of active inflammatory signal transduction in colonic polyps and that these are predominantly in the stroma. In the case of NF-kappaB this coincides with the cellular localisation of COX-2. These results support evidence that NSAIDs may act through effects on stromal cells rather than epithelial cells.

Adenomatous Polyps↗
PubMed2001

Leptin is a growth factor for colonic epithelial cells.

BACKGROUND AND AIMS: Obesity increases the risk of colon cancer, whereas physical activity reduces the risk. Plasma levels of leptin increase in proportion to the level of obesity and are reduced by physical activity. Leptin acts as a growth factor for several cell types and thus may provide a biological explanation for the observed epidemiological risk factors. The aim of this study was to investigate whether leptin is a growth factor for colonic epithelial cells. METHODS: The presence of the leptin receptor in human colon cancer cell lines was assessed using reverse-transcription polymerase chain reaction and immunoblotting, and its presence in human colonic tissue was assessed by immunohistochemistry. The effects of leptin in vitro on HT29 cells were assessed by assessing p42/44 mitogen-activated protein kinase phosphorylation, thymidine incorporation, and cell numbers and in vivo in C57BL/6 mice by colonic bromodeoxyuridine incorporation. RESULTS: The leptin receptor is expressed in human colon cancer cell lines and human colonic tissue. Stimulation with leptin leads to phosphorylation of p42/44 mitogen-activated protein kinase and increases proliferation in vitro and in vivo. CONCLUSIONS: Leptin is a growth factor in colonic epithelial cells and one that may provide a biological explanation for the observed associations between obesity, physical activity, and colon cancer.

Adenocarcinoma↗
PubMed2001

Sonic hedgehog regulates gastric gland morphogenesis in man and mouse.

BACKGROUND & AIMS: Gastric epithelial renewal is an asymmetric process. A stem cell located halfway up the tubular unit gives rise to both a basal gland region and a luminal pit compartment, but the mechanisms responsible for the maintenance of this asymmetry are obscure. We investigated whether Sonic hedgehog (Shh), an established polarizing signal protein during development, is expressed and functional in the adult human and murine stomach. METHODS: Expression of Shh and putative transcriptional targets was investigated using immunoblot and immunohistochemistry. Mice were treated with the Shh inhibitor cyclopamine and examined for expression levels of Shh targets and proliferation of gastric epithelial cells. RESULTS: Shh was expressed in the stomach. In cyclopamine-treated mice, we observed decreased expression of HNF3beta, Islet (Isl)-1 and BMP4, 3 putative Shh target genes. Inhibition of Shh markedly enhanced gastric epithelial proliferation and affected the cell cycle of gastric epithelial gland cells, whereas pit cells remained unaffected. CONCLUSIONS: Shh controls the expression of at least 3 factors important for epithelial differentiation and is a negative regulator of gastric gland cell proliferation. Shh is a candidate polarizing signal in the maintenance of gastric pit-gland asymmetry.

Age Factors↗
PubMed2001

Can cardiotocography prior to induction of labour predict obstetric intervention? A pilot study.

We set out to study the value of cardiotocography (CTG) performed before induction of labour for prolonged pregnancy in predicting obstetric intervention. This was a prospective observational study, based in a district general hospital. We studied 100 consecutive patients who underwent induction of labour for prolonged pregnancy. Cardiotocographs (CTGs) were performed before induction of labour on the tenth day after the estimated date of confinement (290 days). The CTGs were then reviewed without knowledge of the outcome of the induction of labour. Obstetric outcomes were then compared. The main outcome measures were the intrapartum presence or absence of meconium stained liquor and the necessity for obstetric intervention. Ninety per cent of CTGs were normal. There was no difference found between the two groups for operative delivery or the presence of meconium liquor. Caesarean section was more likely in the group with an abnormal CTG before induction of labour, but the possibility of this being due to chance is high in this study. There was one case of undiagnosed growth restriction in the abnormal CTG group. These results may be due to a true lack of difference in obstetric intervention between women with normal or abnormal CTGs prior to induction of labour or more probably an inability to detect a difference in our small study. These baseline data allow the calculations necessary for a more substantive trial.

Journal Article↗
PubMed2000

Pituitary adenylate cyclase-activating polypeptide innervation of the mudpuppy cardiac ganglion.

The presence and potential origin of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) was determined in cardiac ganglia of the mudpuppy, Necturus maculosus. Although PACAP has been implicated in the regulation of cardiac function in several mammalian species, the presence of this peptide in the autonomic nervous system (ANS) of other species is unclear. Thus, this study is the first to characterize this highly conserved peptide in the ANS of a non-mammalian species. PACAP-immunoreactivity was observed in nerve fibers throughout the mudpuppy cardiac ganglia and often was co-localized with the sensory neuropeptides substance P and calcitonin gene-related peptide. Removal of all extrinsic inputs to the ganglia by organ culture eliminated PACAP-immunoreactivity in the cardiac ganglia, whereas bilateral vagotomies only partially reduced PACAP-labeling. PACAP-immunoreactive neurons were observed in both high thoracic dorsal root ganglia and in vagal sensory ganglia. While no PACAP-positive neurons were observed in caudal medulla brainstem regions, PACAP-containing nerve fibers were found in the region of the nucleus solitarius. These results suggest that, in the mudpuppy, PACAP is found primarily in visceral afferent fibers, originating from cells in either the dorsal root ganglia or vagal sensory ganglia. Based on their anatomic localization, these afferent fibers may function to transmit important sensory information to cardiovascular centers in the brain as well as serving as local reflex inputs to modulate postganglionic parasympathetic output within the cardiac ganglion itself.

Afferent Pathways↗
PubMed2000

Leptin signaling in human peripheral blood mononuclear cells, activation of p38 and p42/44 mitogen-activated protein (MAP) kinase and p70 S6 kinase.

The adipocyte-derived hormone leptin plays an important role as a relayer of nutritional status to several organ systems. Evidence is accumulating that leptin plays an important role in the adequate functioning and maintenance of the immune system. Here we show that leptin induces sustained phosphorylation of p38 MAP kinase in human peripheral blood mononuclear cells (PBMCs). We show furthermore that leptin induces two routes to phosphorylation of the 40S ribosomal protein S6, one is activation of the p90 ribosomal S6 kinase (RSK) via the MEK/p42/p44 MAP kinase pathway, the other is via activation of p70 S6 kinase. Thus, these results give new insight in the mechanism that underlies the immunomodulatory effects of leptin.

Culture Media, Serum-Free↗
PubMed2000

PACAP peptides modulate guinea pig cardiac neuron membrane excitability and neuropeptide expression.

Morphological studies identified PACAP-immunoreactive nerve fibers in dense pericellular arrangements around virtually every cholinergic parasympathetic neuron of guinea pig cardiac ganglia; all postganglionic cardiac neurons expressed membrane-associated PAC1 receptor protein. Characterization of the alternative splice variants established predominant expression of the PAC1(very short) receptor transcript containing neither HIP nor HOP exons. PACAP depolarized cardiac neurons and increased membrane excitability; the excitability resulted from neither altered action potential properties nor inhibition of IM. Treatment of cardiac ganglia explants with PACAP significantly reduced the numbers of cholinergic neurons coexpressing somatostatin immunoreactivity, which did not appear to be correlated with prosomatostatin mRNA expression. The PACAP-mediated decrease in somatostatin immunoreactive neurons required calcium influx through L-type calcium channels and activation of adenylyl cyclase, whereas activation of phospholipase C or protein kinase A was not required. These observations indicate that PACAP through the PAC1 receptors elicits complex actions on guinea pig parasympathetic cardiac ganglia neurons, including modulation of membrane ion conductances and modulation of neuropeptide expression.

Animals↗
PubMed1999

Role of mitochondrial dysfunction in the Ca2+-induced decline of transmitter release at K+-depolarized motor neuron terminals.

The present study tested whether a Ca2+-induced disruption of mitochondrial function was responsible for the decline in miniature endplate current (MEPC) frequency that occurs with nerve-muscle preparations maintained in a 35 mM potassium propionate (35 mM KP) solution containing elevated calcium. When the 35 mM KP contained control Ca2+ (1 mM), the MEPC frequency increased and remained elevated for many hours, and the mitochondria within twitch motor neuron terminals were similar in appearance to those in unstimulated terminals. All nerve terminals accumulated FM1-43 when the dye was present for the final 6 min of a 300-min exposure to 35 mM KP with control Ca2+. In contrast, when Ca2+ was increased to 3.6 mM in the 35 mM KP solution, the MEPC frequency initially reached frequencies >350 s-1 but then gradually fell approaching frequencies <50 s-1. A progressive swelling and eventual distortion of mitochondria within the twitch motor neuron terminals occurred during prolonged exposure to 35 mM KP with elevated Ca2+. After approximately 300 min in 35 mM KP with elevated Ca2+, only 58% of the twitch terminals accumulated FM1-43. The decline in MEPC frequency in 35 mM KP with elevated Ca2+ was less when 15 mM glucose was present or when preparations were pretreated with 10 microM oligomycin and then bathed in the 35 mM KP with glucose. When glucose was present, with or without oligomycin pretreatment, a greater percentage of twitch terminals accumulated FM1-43. However, the mitochondria in these preparations were still greatly swollen and distorted. We propose that prolonged depolarization of twitch motor neuron terminals by 35 mM KP with elevated Ca2+ produced a Ca2+-induced decrease in mitochondrial ATP production. Under these conditions, the cytosolic ATP/ADP ratio was decreased thereby compromising both transmitter release and refilling of recycled synaptic vesicles. The addition of glucose stimulated glycolysis which contributed to the maintenance of required ATP levels.

Animals↗
PubMed1998

Mechanisms of pituitary adenylate cyclase activating polypeptide (PACAP)-induced depolarization of sympathetic superior cervical ganglion (SCG) neurons.

Our understanding of PACAP expression and regulation of sympathetic neuronal function has been augmented considerably over the last few years. Among the three major VIP/PACAP receptor subtypes, the SCG appears to express preferentially one particular variant of the PACAP-selective PACAP1 receptor coupled to multiple intracellular signaling cascades. The in situ histochemical hybridization and immunocytochemical studies of PACAP1 receptor mRNA and protein are in good agreement; nearly all of the SCG neurons express the PACAP-selective receptor, suggesting that most of the sympathetic neurons are under PACAP neuromodulation. In accord with that possibility, several independent studies have now demonstrated PACAP peptide expression in the IML sympathetic preganglionic neurons and fibers, including those projecting to the SCG, further emphasizing the significance of PACAP peptides as a preganglionic noncholinergic mediator of sympathetic function. Given the high potency of PACAP on any of a number of cellular responses, the functional relevance of PACAP peptides on SCG neurons is considerable. We have previously demonstrated the potency and efficacy of both PACAP27 and PACAP38 on sympathetic neuron neurotransmitter/neuropeptide production and secretion; the ability of these peptides to stimulate neuronal second messenger activation was also in the nanomolar range. These results are congruous with our current electrophysiological studies, which were driven to further define the dynamic sympathetic responses to PACAP. In line with the morphological studies, for example, more than 90% of the sympathetic neurons responded to PACAP. In agreement with previous neuropharmacological data, the PACAP-induced depolarizations were elicited at physiologically relevant peptide concentrations at high affinity PACAP-selective receptors. The effects were direct and the alterations in postganglionic neuronal membrane properties appeared to be mediated by several ionic mechanisms. If these studies were analogous to pieces in a puzzle to understand the effects of PACAP in sympathetic development and function, the picture of late has been more completely assembled. But several important challenges still remain. What are the signal transduction mechanisms that mediate the PACAP-induced changes in sympathetic membrane properties? How do the resulting alterations impact the acute and more long-term responses of sympathetic neurons? Does the coupling of PACAP1 receptors to intracellular signaling pathways differ during development, resulting in a transition from the neurotrophic properties of PACAP in neuroblasts to neuromodulatory roles of the peptides in postmitotic neurons? By looking at these issues in one distinct neuronal system, we enlarge our understanding and appreciation of peptides, and PACAP in particular, in the molecular and cellular events guiding neuronal development, function, and plasticity.

Animals↗
PubMed1998

Pituitary adenylate cyclase-activating polypeptide expression and modulation of neuronal excitability in guinea pig cardiac ganglia.

Cardiac output is regulated by the coordinate interactions of stimulatory sympathetic and inhibitory parasympathetic signals. Intracardiac parasympathetic ganglia are integrative centers of cardiac regulation, and modulation of the parasympathetic drive on the heart is accomplished by altering intrinsic cardiac ganglion neuron excitability. The pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) family of peptides modulates cardiac function, and in guinea pig heart, PACAP appears to act directly on intrinsic parasympathetic cardiac ganglia neurons through PACAP-selective receptors. A multidisciplinary project tested whether cardiac PACAP peptides act through PACAP-selective receptors as excitatory neuromodulators amplifying the parasympathetic inhibition from guinea pig cardiac ganglia. The in vivo sources of regulatory PACAP peptides were localized immunocytochemically to neuronal fibers and a subpopulation of intrinsic postganglionic cardiac neurons. RT-PCR confirmed that cardiac ganglia expressed proPACAP transcripts and have PACAP peptide biosynthetic capabilities. Messenger RNA encoding PACAP-selective PAC1 receptor isoforms were also present in cardiac ganglia. Alternative splicing of PAC1 receptor transcripts produced predominant expression of the very short variant with neither HIP nor HOP cassettes; lower levels of the PAC1HOP2 receptor mRNA were present. Almost all of the parasympathetic neurons expressed membrane-associated PAC1 receptor proteins, localized immunocytochemically, which correlated with the population of cells that responded physiologically to PACAP peptides. PACAP depolarized cardiac ganglia neurons and increased neuronal membrane excitability. The rank order of peptide potency on membrane excitability in response to depolarizing currents was PACAP27>PACAP38>VIP. The PACAP-induced increase in excitability was not a function of membrane depolarization nor was it caused by alterations in action potential configuration. These results support roles for PACAP peptides as integrative modulators amplifying, through PACAP-selective receptors, the parasympathetic cardiac ganglia inhibition of cardiac output.

Adenylyl Cyclases↗
PubMed1998

Expression and physiological actions of neuropeptide Y in guinea pig parasympathetic cardiac ganglia.

Guinea pig atrial whole mount preparations containing the parasympathetic cardiac ganglia were used to establish the expression, distribution and actions of neuropeptide Y (NPY) in atrial tissues. NPY-immunoreactive fibers densely innervated the atrial myocardium and blood vessels. Fibers containing NPY also innervated intrinsic parasympathetic cardiac neurons. Four percent of the cardiac neurons, identified using microtubule associated protein-2 antiserum, were NPY-positive. An endogenous source of NPY was confirmed with reverse transcription PCR which demonstrated the presence of proNPY mRNA. Sixty percent of the parasympathetic cardiac neurons were hyperpolarized by local application of NPY. NPY also decreased the amplitude and duration of the action potential after hyperpolarization in 60% of the neurons and decreased the fast excitatory postsynaptic potential in about 50% of the cells. These observations indicate that NPY is anatomically positioned to directly alter the output of the parasympathetic cardiac ganglia either by hyperpolarizing the cardiac neurons or by decreasing the fast synaptic input which drives individual neurons.

Animals↗
PubMed1997

Tachykinin-induced activation of non-specific cation conductance via NK3 neurokinin receptors in guinea-pig intracardiac neurones.

1. Whole mount preparations from guinea-pig hearts were used to characterize the receptors and ionic mechanisms mediating the substance P (SP)-induced depolarization of parasympathetic postganglionic neurones of the cardiac ganglion. 2. Measurement of the amplitude of depolarization in response to superfusion of different tachykinin agonists (neurokinins A (NKA) and B (NKB), SP, and senktide) gave a rank-order potency of NKB = senktide > NKA > SP, indicating involvement of an NK3 receptor. The use of the selective tachykinin receptor antagonists SR 140333, SR 48986, and SR 142801 demonstrated that only the NK3 receptor antagonist SR 142801 inhibited the SP-induced depolarization. 3. The SP-induced depolarization was not inhibited by Ba2+, TEA, or niflumic acid, or altered by reduced Cl- solutions, but was attenuated in reduced Na+ solutions. Single electrode voltage clamp studies demonstrated that the SP-induced inward current increased in amplitude at more negative potentials, had a reversal potential of approximately 0 mV, and was reduced in amplitude in reduced Na+ solutions. 4. We conclude that the SP-induced depolarization in guinea-pig postganglionic parasympathetic neurones of the cardiac ganglion is due to NK3-mediated activation of a non-selective cation conductance.

Animals↗
PubMed1997

Transmitter release differs at snake twitch and tonic endplates during potassium-induced nerve terminal depolarization.

Twitch and tonic muscle fibers of snake skeletal muscle differ in their synpatic as well as mechanical properties. These experiments were aimed at detemining the basis of the difference in vesicular release properties of nerve terminals at twitch and tonic endplates. Miniature endplate currents (MEPCs) were recorded from voltage-clamped garter snake muscle fibers depolarized by high K+ in either a control Ca2+ or high-Ca2+ solution. MEPC frequency increased at twitch and tonic endplates and remained elevated for 8 h during depolarization in control Ca2+. At twitch endplates depolarized in the presence of high Ca2+, an increase in MEPC frequency was followed by a progressive decline. In contrast, MEPC frequency remained elevated in high Ca2+ at tonic endplates. The observed decrease in MEPC frequency at depolarized twitch endplates in high Ca2+ was not a function of the level of depolarization or initial MEPC frequency, nor was it due to a reduction in MEPC amplitude and loss of MEPCs in baseline noise. An optical assay of presynaptic function in which the activity-dependent dye FM1-43 was used confirmed that quantal releases differs at twitch and tonic endplates. Most twitch nerve terminals were labeled by FM1-43 during prolonged depolarization with control Ca2+ or after brief depolarization with high Ca2+. In contrast, the number of twitch nerve terminals and the degree to which they were stained was greatly reduced after prolonged exposure to high K+ and high Ca2+, whereas depolarized tonic endplates were well stained by FM1-43 during brief and prolonged exposure to high Ca2+. FM1-43 staining also revealed variable levels of quantal release between individual boutons at twitch endplates after prolonged depolarization in high-Ca2+ solution. The observed reduction in presynaptic function at twitch nerve terminals after prolonged depolarization in high-Ca2+ solution was reversible and therefore not due to irreversible damage to terminal boutons. MEPC frequency increased at both twitch and tonic endplates when either Sr2+ or Ba2+ was substituted for high Ca2+ during K(+)-induced depolarization. Over time, in Sr2+ or Ba2+ solutions, MEPC frequency remained elevated at tonic endplates but declined at twitch endplates with a time course similar to that observed in high Ca2+. MEPC amplitudes at both endplates remained constant. We conclude that the regulation of quantal release differs in nerve terminals innervating twitch and tonic endplates and postulate that differential intraterminal accumulation of Ca2+ may underlie the observed difference in presynaptic function.

Animals↗