Mark J. Drinkhill

Stimulation of ovine placental transport of calcium and magnesium by mid-molecule fragments of human parathyroid hormone-related protein

Perfusion in situ of the placenta of intact or previously parathyroidectomized fetal lambs has been used to assess the ability of three mid‐molecule fragments of the human parathyroid hormone‐related protein (PTHrP) molecule to stimulate the placental transport of calcium and magnesium. PTHrP(67‐86 amide) was most effective but some activity was also shown by PTHrP(75‐86 amide) and by PTHrP (75‐84) in decreasing order. This placental action of PTHrP(67‐86 amide) was rapid and could be observed using the placenta from an intact fetus, whereas it was necessary to use the placenta from a previously parathyroidectomized fetus to demonstrate stimulation of placental calcium transport by PTHrP(1‐84). PTHrP(67‐86 amide) may resemble the molecule that activates the placental calcium pump.

Change in endothelial function in mesenteric arteries of sprague‐dawley rats fed a high salt diet

A high salt diet in some species results in elevated arterial blood pressure and alterations in vascular smooth muscle responses to agonists. Weanling male Sprague‐Dawley rats were given either a high salt diet containing 8 % or a low salt diet of 0.4 % sodium chloride for a period of 4 weeks. At the end of the feeding period, tail systolic pressure was higher in the high salt than in low salt rats. The rats were then killed and the intestines removed. Vascular smooth muscle (VSM) responses were estimated from the changes in lumenal diameter of pressurised second order mesenteric resistance arteries. High salt diet resulted in enhanced VSM responses to noradrenaline. The vessels dilated in response both to acetylcholine and to sodium nitroprusside and the responses were similar in vessels from both high and low salt rats. However, vessels from high salt rats were resistant to the blocking of endothelium derived nitric oxide (EDNO) with L‐NAME and the responses were instead abolished by blocking endothelium derived hyperpolarising factor (EDHF) with apamin and charybdotoxin. These results show that in Sprague‐Dawley rats, a high salt diet enhances the vasoconstriction in response to noradrenaline. The vasodilatory responses to acetylcholine were not significantly changed. However, they appeared to be mediated mainly by EDHF rather than by EDNO as in the low salt animals.

The autonomic nervous system at high altitude

The effects of hypobaric hypoxia in visitors depend not only on the actual elevation but also on the rate of ascent. Sympathetic activity increases and there are increases in blood pressure and heart rate. Pulmonary vasoconstriction leads to pulmonary hypertension, particularly during exercise. The sympathetic excitation results from hypoxia, partly through chemoreceptor reflexes and partly through altered baroreceptor function. High pulmonary arterial pressures may also cause reflex systemic vasoconstriction. Most permanent high altitude dwellers show excellent adaptation although there are differences between populations in the extent of the ventilatory drive and the erythropoiesis. Some altitude dwellers, particularly Andeans, may develop chronic mountain sickness, the most prominent characteristic of which being excessive polycythaemia. Excessive hypoxia due to peripheral chemoreceptor dysfunction has been suggested as a cause. The hyperviscous blood leads to pulmonary hypertension, symptoms of cerebral hypoperfusion, and eventually right heart failure and death.

Angiotensin II type-1 receptor activation in the adult heart causes blood pressure-independent hypertrophy and cardiac dysfunction

AIMS Sustained hypertension leads to cardiac hypertrophy that can progress, through pathological remodelling, to heart failure. Abnormality of the renin-angiotensin system (RAS) has been strongly implicated in this process. Although hypertrophy in human is an established risk factor independent of blood pressure (BP), separation of remodelling in response to local cues within the differentiated myocardium from that related to pressure overload is unresolved. This study aimed to clarify the role of local RAS activity, specifically in the adult heart, in modulating cardiac hypertrophy and pathological remodelling. METHODS AND RESULTS Transgenic mice with inducible cardiomyocyte-specific expression of a wild-type or N111G mutant form of the human angiotensin II (Ang II) type-1 receptor (hAT1R) were generated. The wild-type receptor is primarily stimulated by Ang II. In contrast, the N111G receptor can also be fully stimulated by the Ang II derivative, Ang IV, at levels that do not stimulate the wild-type receptor. The unique properties of these models were used to investigate the myocardial growth, remodelling and functional responses to hAT1R stimulation, specifically in adult cardiomyocytes, under normal conditions and following Ang IV infusion. Low-level expression of wild-type or N111G hAT1R at the cardiomyocyte membrane, from the onset of adolescence, induced enhanced myocyte growth and associated cardiac hypertrophy in the adult. This was not associated with change in resting BP or heart rate, measured by longitudinal telemetric analysis, and did not progress to pathological remodelling or heart failure. However, selective activation of cardiomyocyte-specific N111G receptors by Ang IV peptide infusion induced adverse ventricular remodelling within 4 weeks. This was characterized by increased interstitial fibrosis, dilatation of the left ventricle, and impaired cardiac function. CONCLUSION Low-level local AT1R activity in differentiated myocardium causes compensated cardiac hypertrophy, that is, increased myocardial mass but with the retention of normal function, whereas short-term increased stimulation induces cardiac dysfunction with dilatation, reduced ejection fraction, and increased fibrosis in the absence of change in systemic BP.

Cardiovascular adjustments for life at high altitude.

The effects of hypobaric hypoxia in visitors depend not only on the actual elevation but also on the rate of ascent. There are increases in sympathetic activity resulting in increases in systemic vascular resistance, blood pressure and heart rate. Pulmonary vasoconstriction leads to pulmonary hypertension, particularly during exercise. The sympathetic excitation results from hypoxia, partly through chemoreceptor reflexes and partly through altered baroreceptor function. Systemic vasoconstriction may also occur as a reflex response to the high pulmonary arterial pressures. Many communities live permanently at high altitude and most dwellers show excellent adaptation although there are differences between populations in the extent of the ventilatory drive and the erythropoiesis. Despite living all their lives at altitude, some dwellers, particularly Andeans, may develop a maladaptation syndrome known as chronic mountain sickness. The most prominent characteristic of this is excessive polycythaemia, the cause of which has been attributed to peripheral chemoreceptor dysfunction. The hyperviscous blood leads to pulmonary hypertension, symptoms of cerebral hypoperfusion, and eventually right heart failure and death.

Arrhythmogenic substrate in hearts of rats with monocrotaline-induced pulmonary hypertension and right ventricular hypertrophy

Mechanisms associated with right ventricular (RV) hypertension and arrhythmias are less understood than those in the left ventricle (LV). The aim of our study was to investigate whether and by what mechanisms a proarrhythmic substrate exists in a rat model of RV hypertension and hypertrophy. Rats were injected with monocrotaline (MCT; 60 mg/kg) to induce pulmonary artery hypertension or with saline (CON). Myocardial levels of mRNA for genes expressing ion channels were measured by real-time RT-PCR. Monophasic action potential duration (MAPD) was recorded in isolated Langendorff-perfused hearts. MAPD restitution was measured, and arrhythmias were induced by burst stimulation. Twenty-two to twenty-six days after treatment, MCT animals had RV hypertension, hypertrophy, and decreased ejection fractions compared with CON. A greater proportion of MCT hearts developed sustained ventricular tachycardias/fibrillation (0.83 MCT vs. 0.14 CON). MAPD was prolonged in RV and less so in the LV of MCT hearts. There were decreased levels of mRNA for K(+) channels. Restitution curves of MCT RV were steeper than CON RV or either LV. Dispersion of MAPD was greater in MCT hearts and was dependent on stimulation frequency. Computer simulations based on ion channel gene expression closely predicted experimental changes in MAPD and restitution. We have identified a proarrhythmic substrate in the hearts of MCT-treated rats. We conclude that steeper RV electrical restitution and rate-dependant RV-LV action potential duration dispersion may be contributing mechanisms and be implicated in the generation of arrhythmias associated with in RV hypertension and hypertrophy.

Cardiac arrhythmia mechanisms in rats with heart failure induced by pulmonary hypertension

Pulmonary hypertension provokes right heart failure and arrhythmias. Better understanding of the mechanisms underlying these arrhythmias is needed to facilitate new therapeutic approaches for the hypertensive, failing right ventricle (RV). The aim of our study was to identify the mechanisms generating arrhythmias in a model of RV failure induced by pulmonary hypertension. Rats were injected with monocrotaline to induce either RV hypertrophy or failure or with saline (control). ECGs were measured in conscious, unrestrained animals by telemetry. In isolated hearts, electrical activity was measured by optical mapping and myofiber orientation by diffusion tensor-MRI. Sarcoplasmic reticular Ca(2+) handling was studied in single myocytes. Compared with control animals, the T-wave of the ECG was prolonged and in three of seven heart failure animals, prominent T-wave alternans occurred. Discordant action potential (AP) alternans occurred in isolated failing hearts and Ca(2+) transient alternans in failing myocytes. In failing hearts, AP duration and dispersion were increased; conduction velocity and AP restitution were steeper. The latter was intrinsic to failing single myocytes. Failing hearts had greater fiber angle disarray; this correlated with AP duration. Failing myocytes had reduced sarco(endo)plasmic reticular Ca(2+)-ATPase activity, increased sarcoplasmic reticular Ca(2+)-release fraction, and increased Ca(2+) spark leak. In hypertrophied hearts and myocytes, dysfunctional adaptation had begun, but alternans did not develop. We conclude that increased electrical and structural heterogeneity and dysfunctional sarcoplasmic reticular Ca(2+) handling increased the probability of alternans, a proarrhythmic predictor of sudden cardiac death. These mechanisms are potential therapeutic targets for the correction of arrhythmias in hypertensive, failing RVs.

Alternating Hemiplegia of Childhood-Related Neural and Behavioural Phenotypes in Na+,K+-ATPase α3 Missense Mutant Mice

Missense mutations in ATP1A3 encoding Na+,K+-ATPase α3 have been identified as the primary cause of alternating hemiplegia of childhood (AHC), a motor disorder with onset typically before the age of 6 months. Affected children tend to be of short stature and can also have epilepsy, ataxia and learning disability. The Na+,K+-ATPase has a well-known role in maintaining electrochemical gradients across cell membranes, but our understanding of how the mutations cause AHC is limited. Myshkin mutant mice carry an amino acid change (I810N) that affects the same position in Na+,K+-ATPase α3 as I810S found in AHC. Using molecular modelling, we show that the Myshkin and AHC mutations display similarly severe structural impacts on Na+,K+-ATPase α3, including upon the K+ pore and predicted K+ binding sites. Behavioural analysis of Myshkin mice revealed phenotypic abnormalities similar to symptoms of AHC, including motor dysfunction and cognitive impairment. 2-DG imaging of Myshkin mice identified compromised thalamocortical functioning that includes a deficit in frontal cortex functioning (hypofrontality), directly mirroring that reported in AHC, along with reduced thalamocortical functional connectivity. Our results thus provide validation for missense mutations in Na+,K+-ATPase α3 as a cause of AHC, and highlight Myshkin mice as a starting point for the exploration of disease mechanisms and novel treatments in AHC.

Central nucleus of amygdala projections to rostral ventrolateral medulla neurones activated by decreased blood pressure.

The central nucleus of amygdala (CeA) participates in cardiovascular regulation during emotional behaviour but it has not been established whether any of these effects are mediated through its direct connections to blood pressure‐regulating neurones in the rostral ventrolateral medulla (RVLM). The RVLM contains barosensitive neurones that maintain resting blood pressure via their projections to sympathetic preganglionic neurones in the thoracic spinal cord. In this study on rats, we used combined anterograde neuronal tracing of CeA projections with confocal and electron microscopic immunohistochemical detection of phenylethanolamine‐N‐methyltransferase, the adrenaline‐synthesizing enzyme present in C1 catecholamine neurones of the RVLM, and Fos, the protein product of the c‐fos proto‐oncogene. Fos expression in barosensitive neurones was stimulated by an intravenous infusion of the hypotensive agent sodium nitroprusside. Injection of the tracer biotin dextran amine (10‐kDa form) into the CeA resulted in anterograde labelling of axons and varicosities throughout the RVLM without retrograde labelling of somata in any brain area. With confocal microscopy, presumptive CeA terminals were found in close apposition to adrenergic (phenylethanolamine‐N‐methyltransferase‐immunoreactive) and non‐adrenergic neurones that displayed Fos‐immunoreactive nuclei in response to decreased blood pressure. Electron microscopic analysis confirmed that some labelled terminals of CeA axons made synaptic contact with c‐fos‐activated adrenergic neurones. The results provide evidence that cardiovascular influences elicited from the CeA during stressful events may be mediated, at least in part, via monosynaptic neural projections to barosensitive sympathetic blood pressure‐regulating neurones in the RVLM.

Reflex Responses from the Main Pulmonary Artery and Bifurcation in Anaesthetised Dogs

This study was undertaken to determine the reflex cardiovascular and respiratory responses to discrete stimulation of pulmonary arterial baroreceptors using a preparation in which secondary modulation of responses from other reflexes was prevented. Dogs were anaesthetised with α‐chloralose, artificially ventilated, the chests widely opened and a cardiopulmonary bypass established. The main pulmonary arterial trunk, bifurcation and extrapulmonary arteries as far as the first lobar arteries on each side were vascularly isolated and perfused through the left pulmonary artery and drained via the right artery through a Starling resistance which controlled pulmonary arterial pressure. Pressures distending systemic baroreceptors and reflexogenic regions in the heart were controlled. Reflex vascular responses were assessed from changes in perfusion pressures to a vascularly isolated hind limb and to the remainder of the subdiaphragmatic systemic circulation, both of which were perfused at constant flows. Respiratory responses were assessed from recordings of efferent phrenic nerve activity. Increases in pulmonary arterial pressure consistently evoked increases in both perfusion pressures and in phrenic nerve activity. Both vascular and respiratory responses were obtained when pulmonary arterial pressure was increased to above about 30 mmHg. Responses increased at higher levels of pulmonary arterial pressures. In 13 dogs increases in pulmonary arterial pressure to 45 mmHg increased systemic perfusion pressure by 24 ± 7 mmHg (mean ± S.E.M.) from 162 ± 11 mmHg. Setting carotid sinus pressure at different levels did not influence the vascular response to changes in pulmonary arterial pressure. The presence of a negative intrathoracic pressure of ‐20 mmHg resulted in larger vascular responses being obtained at lower levels of pulmonary arterial pressure. This indicates that the reflex may be more effective in the intact closed‐chest animal. These results demonstrate that stimulation of pulmonary arterial baroreceptors evokes a pressor reflex and augments respiratory drive. This reflex is likely to be elicited in circumstances where pulmonary arterial pressure increases and the negative excursions of intrathoracic pressure become greater. They are likely, therefore, to be involved in the cardio‐respiratory response to exercise as well as in pathological states such as pulmonary hypertension or restrictive or obstructive lung disease.