Impact of prenatal and postnatal maternal environment on nephron endowment, renal function and blood pressure in the Lewis polycystic kidney rat.

Maternal insufficiency during fetal development can have long-lasting effects on the offspring, most notably on nephron endowment. In polycystic kidney disease (PKD), variability in severity of disease is observed and maternal environment may be a modifying factor. In this study, we first established that in a rodent model of PKD, the Lewis polycystic kidney (LPK) rat's nephron numbers are 25% lower compared with wildtype animals. We then investigated the effects of prenatal and postnatal maternal environment on phenotype and nephron number. LPK pups born from and raised by homozygous LPK dams (control) were compared with LPK pups cross-fostered onto heterozygous LPK dams to improve postnatal environment; with LPK pups born from and raised by heterozygous LPK dams to improve both prenatal and postnatal environment and with LPK pups born from and raised by Wistar Kyoto-LPK heterozygous dams to improve both prenatal and postnatal environment on a different genetic background. Improvement in both prenatal and postnatal environment improved postnatal growth, renal function and reduced blood pressure, most notably in animals with different genetic background. Animals with improved postnatal environment only showed improved growth and blood pressure, but to a lesser extent. All intervention groups showed increased nephron number compared with control LPK. In summary, prenatal and postnatal environment had significant effect in delaying progression and reducing severity of PKD, including nephron endowment.

Uraemia: an unrecognized driver of central neurohumoral dysfunction in chronic kidney disease?

Chronic kidney disease (CKD) carries a large cardiovascular burden in part due to hypertension and neurohumoral dysfunction - manifesting as sympathetic overactivity, baroreflex dysfunction and chronically elevated circulating vasopressin. Alterations within the central nervous system (CNS) are necessary for the expression of neurohumoral dysfunction in CKD; however, the underlying mechanisms are poorly defined. Uraemic toxins are a diverse group of compounds that accumulate as a direct result of renal disease and drive dysfunction in multiple organs, including the brain. Intensive haemodialysis improves both sympathetic overactivity and cardiac baroreflex sensitivity in renal failure patients, indicating that uraemic toxins participate in the maintenance of autonomic dysfunction in CKD. In rodents exposed to uraemia, immediate early gene expression analysis suggests upregulated activity of not only pre-sympathetic but also vasopressin-secretory nuclei. We outline several potential mechanisms by which uraemia might drive neurohumoral dysfunction in CKD. These include superoxide-dependent effects on neural activity, depletion of nitric oxide and induction of low-grade systemic inflammation. Recent evidence has highlighted superoxide production as an intermediate for the depolarizing effect of some uraemic toxins on neuronal cells. We provide preliminary data indicating augmented superoxide production within the hypothalamic paraventricular nucleus in the Lewis polycystic kidney rat, which might be important for mediating the neurohumoral dysfunction exhibited in this CKD model. We speculate that the uraemic state might serve to sensitize the central actions of other sympathoexcitatory factors, including renal afferent nerve inputs to the CNS and angiotensin II, by way of recruiting convergent superoxide-dependent and pro-inflammatory pathways.

Progressive vascular remodelling, endothelial dysfunction and stiffness in mesenteric resistance arteries in a rodent model of chronic kidney disease.

Chronic kidney disease (CKD) and hypertension are co-morbid conditions both associated with altered resistance artery structure, biomechanics and function. We examined these characteristics in mesenteric artery together with renal function and systolic blood pressure (SBP) changes in the Lewis polycystic kidney (LPK) rat model of CKD. Animals were studied at early (6-weeks), intermediate (12-weeks), and late (18-weeks) time-points (n=21), relative to age-matched Lewis controls (n=29). At 12 and 18-weeks, LPK arteries exhibited eutrophic and hypertrophic inward remodelling characterised by thickened medial smooth muscle, decreased lumen diameter, and unchanged or increased media cross-sectional area, respectively. At these later time points, endothelium-dependent vasorelaxation was also compromised, associated with impaired endothelium-dependent hyperpolarisation and reduced nitric oxide synthase activity. Stiffness, elastic-modulus/stress slopes and collagen/elastin ratios were increased in 6 and 18-week-old-LPK, in contrast to greater arterial compliance at 12weeks. Multiple linear regression analysis highlighted SBP as the main predictor of wall-lumen ratio (r=0.536, P<0.001 n=46 pairs). Concentration-response curves revealed increased sensitivity to phenylephrine but not potassium chloride in 18-week-LPK. Our results indicate that impairment in LPK resistance vasculature is evident at 6weeks, and worsens with hypertension and progression of renal disease.

Trait perfectionism strengthens the negative effects of moral stressors occurring in veterinary practice.

OBJECTIVE: Moral challenges are a unique class of workplace stressor where behaviours violate one's personal moral beliefs regarding how things should be done or one's perceived obligations. Morally challenging stressors exist in many workplaces and at times can transform into marked emotional distress, referred to as moral distress. In this study we investigated the degree to which morally significant stressors are related to psychological distress and resilience in a sample of Australian veterinarians. Further, we explored the role of trait perfectionism in strengthening the relationship between exposure to morally significant stressors and psychological distress. Trait perfectionism is the tendency to have very high and rigid standards for the self and/or others and is often implicated in the experience of psychological distress. METHODS: A cross-sectional online survey sampled 540 Australian-registered veterinarians (64.2% female), ranging in age from 23 to 74 years. RESULTS: Although morally significant stressors were related to increases in milder expressions of distress, they did not appear to be associated with more severe decrements in psychological wellbeing. Rather, it was the combination of these triggering stressor events and trait perfectionism that appeared to create the vulnerability to moral stressors. CONCLUSION: The findings suggest that trait perfectionism is an individual difference that enhances vulnerability to the risk of greater distress in response to morally challenging events in veterinary practice. The implications of these findings and directions for further research are discussed.

Improved Survival with Anti-VEGF Therapy in the Treatment of Unresectable Appendiceal Epithelial Neoplasms.

BACKGROUND: Currently, cytoreductive surgery and hyperthermic intraperitoneal chemotherapy are accepted treatments for surgically resectable appendiceal epithelial neoplasms. However, for nonsurgical candidates, systemic treatment may be considered. The purpose of this analysis was to determine the benefit of biologic therapy (anti-vascular endothelial growth factor and anti-epidermal growth factor receptor) in addition to systemic chemotherapy in this select patient population. METHODS: The MD Anderson Cancer Center tumor registry was retrospectively reviewed for systemic treatment-naive appendiceal epithelial neoplasm patients registered between January 2000 to July 2007 for prior cytoreductive surgery and hyperthermic intraperitoneal chemotherapy status, histologic grade, signet ring pathology, systemic chemotherapy, biologic therapy, tumor markers (carcinoembryonic antigen, carbohydrate antigen [CA] 125, and/or CA19-9), progression-free survival (PFS), overall survival (OS), and disease control rate. Kaplan-Meier method, log-rank, and Cox proportional hazard regression models were used for statistical analysis. RESULTS: A total of 353 patients were identified; 130 patients met the inclusion criteria. Fifty-nine patients received biologic therapy. The use of the anti-vascular endothelial growth factor (VEGF) agent bevacizumab improved both OS (42 months vs. 76 months, hazard ratio 0.49 [95 % confidence interval 0.25-0.94] P = 0.03) and PFS (4 months vs. 9 months, hazard ratio 0.69 [95 % confidence interval 0.47-0.995], P = 0.047) for all histologic subtypes. Moderately differentiated tumors had an improved PFS relative to well-differentiated tumors, 9 months versus 3 months (P = 0.05). CONCLUSIONS: Bevacizumab in combination with chemotherapy appears to play a role in surgically unresectable appendiceal epithelial neoplasm patients, with an improvement in PFS and OS. Anti-VEGF agents should be strongly considered in the management of patients with higher-grade appendiceal epithelial neoplasms who are suboptimal candidates for surgical resection.

The influence of spasmolytic agents on heart rate variability and gastrointestinal motility in normal horses.

The effects of hyoscine-N-butylbromide (hyoscine) and propantheline-bromide (propantheline) on heart rate (HR), HR variability (HRV) and gastrointestinal tract (GIT) contractions in the normal horse were determined. Five adult horses had ECG recordings for 180 min after treatment with propantheline (100mg), hyoscine (120 mg) or saline. Both propantheline and hyoscine reduced GIT sounds, with propantheline having a longer duration of effect (≥120 min). Both drugs elevated HR relative to the control baseline period (P<0.05), with the effects of propantheline again being of longer duration. HRV analysis indicated that propantheline suppressed Total Power (P<0.05), and both the high frequency (HF) and low frequency (LF) components of the power spectral analysis for up to 60-90 min post treatment. Hyoscine had no effect on HRV Total Power but reduced the HF component for 30 min after drug injection. Time domain variables correlated with Total Power and HF data (P<0.01). The marked effect of these compounds on parasympathetic control of cardiac and GIT function in normal horses should be taken into consideration when evaluating a clinical response to these agents.

Expression of α1-adrenoceptors on peripheral nociceptive neurons.

The purpose of this study was to determine whether α(1)-adrenoceptors are expressed on primary nociceptive afferents that innervate healthy skin. Skin and dorsal root ganglia were collected from adult male Wistar rats and assessed using fluorescence immunohistochemistry with antibodies directed against α(1)-adrenoceptors alone or in combination with specific labels including myelin basic protein and neurofilament 200 (markers of myelinated nerve fibres), protein gene product 9.5 (a pan-neuronal marker), tyrosine hydroxylase (sympathetic neurons), isolectin B(4) (IB(4): non-peptidergic sensory neurons), calcitonin gene related peptide (CGRP) and transient receptor potential vanilloid receptor 1 (TRPV1) (peptidergic sensory neurons). Double labelling in dorsal root ganglia confirmed the expression of α(1)-adrenoceptors within sub-populations of CGRP, IB(4) and TRPV1 immunoreactive neurons. Myelinated and unmyelinated sensory nerve fibres in the skin expressed α(1)-adrenoceptors whereas sympathetic nerve fibres did not. The expression of α(1)-adrenoceptors on C- and A-delta nociceptive afferent fibres provides a histochemical substrate for direct excitation of these fibres by adrenergic agonists. This may help to explain the mechanism of sensory-sympathetic coupling that sometimes develops on surviving primary nociceptive afferents in neuropathic pain states.

Alternating frequency to increase the response to stimulation from medium voltage electrical stimulation and the effect on objective meat quality.

The use of alternating frequencies during stimulation can increase stimulation response of a medium voltage electrical stimulation unit (MVS) by increasing the rate of pH decline. Various combinations of frequency modulation were tested in experiment 1 to determine the treatment resulting in the greatest stimulation response; the lowest initial pH, fastest rate of pH decline, highest temperature at pH 6 and the highest number of carcasses with a pH of 6 by 25(o)C and the treatment achieving the highest number of carcasses in the pH temp window (temperature at pH 6 between 18-25(o)C). The objective meat quality of these treatments compared to an unstimulated treatment was tested in experiment 2. Modulating the frequency (Hz) across the 6 segmented electrodes of the MVS by 10, 15, 25, 10, 15, 25Hz (Treatment 6, using a pulse width: 2.5ms, current: 1A) resulted in the greatest stimulation response. This treatment may be suitable for abattoirs that hot bone sheepmeat and require fast pH declines to ensure minimal cold shortening of meat. However, this treatment did not result in the tenderer meat despite the higher stimulation response. This treatment may have induced a greater number of contractions overall and therefore a greater pH decline response but resulted in less myofibrillar disruption compared to the other treatments due to a concomitant decreased force of contraction thus reducing potential tenderisation. Maintaining a constant frequency of 15Hz (Treatment 1; pulse width: 2.5ms, current: 1A) resulted in a higher number of carcasses in the pH temp window required (temperature at pH 6 between 18-25(o)C) in part A (P<0.05) and in addition to the higher tenderness levels this treatment may be more appropriate to satisfy the overall demands of abattoirs using these systems. This paper has also demonstrated electrical stimulation results in tenderer meat compared to unstimulated meat even after 30d of ageing (2.53±0.4 compared to 2.85±0.1 for the loin (M. longissimus thoracis et lumborum) (P<0.01) possibly due to a protective benefit of stimulation on meat tenderness. Overall, no detrimental effects of modulating frequency were observed on drip loss or retail colour display despite a greater rate of colour change observed with the modulated frequency treatment and the longer aged product.

Chromogranin a expression in phaeochromocytomas associated with von Hippel-Lindau syndrome and multiple endocrine neoplasia type 2.

Chromogranin A (CGA) is a major secretory protein present in the soluble matrix of chromaffin granules of neuroendocrine cells and tumours, such as phaeochromocytomas. CGA has several functions, some of which may be involved in the distinct phenotypic differences of phaeochromocytomas in patients with von Hippel-Lindau (VHL) syndrome compared to multiple endocrine neoplasia type 2 (MEN 2). In this study, we therefore compared tumour and plasma levels of CGA in patients with phaeochromocytoma associated with the two syndromes. We show that phaeochromocytomas from MEN 2 patients express substantially more CGA than tumours from VHL patients at both the mRNA (3-fold greater) and protein (20-fold) level. We further show that relative to increases in plasma catecholamines, patients with phaeochromocytomas associated with MEN 2 have higher plasma concentrations of CGA than those with tumours in VHL syndrome. These data supplement other observations that phaeochromocytomas in VHL compared to MEN 2 patients express lower amounts of catecholamines and other chromaffin granule cargo, such as chromogranin B and neuropeptide Y. Possibly the differences in tumour CGA expression may contribute to differences in secretory vesicle formation and secretion in the two types of tumours. Alternatively the differences in expression in CGA and other secretory constituents may reflect downregulation of the entire regulated secretory pathway in VHL compared to MEN 2 tumours.

Intrathecal cGMP elicits pressor responses and maintains mean blood pressure during haemorrhage in anaesthetized rats.

The intracellular second messenger, cyclic guanosine monophosphate (cGMP), a soluble guanylate cyclase (GC) product, is a primary mechanism for the transduction of a nitric oxide (NO)-initiated signal in the central nervous system. NO is produced from L-arginine by neuronal nitric oxide synthase (NOS), which is found in sympathetic preganglionic neurons of the intermediolateral cell column. This suggests the possibility that NO is a modulator of sympathetic nervous activity (SNA) through a cGMP-mediated mechanism. The aim of this study was to determine the effects of intrathecally injected membrane-permeant 8-bromo-cGMP and 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of the soluble form of GC, on arterial pressure in urethane anaesthetized (1.4 g kg(-1) I.P.) rats. The effects of intrathecal cGMP and ODQ on haemodynamic responses to haemorrhage were also investigated. Finally, L-arginine, the NO precursor, was also injected intrathecally, alone and in the presence of ODQ. Baseline mean arterial pressure (MAP) increased significantly after intrathecal 8-Br-cGMP injection (10 microl, 1, 3, 10, 30, 100 microm). A dose-effect relationship (1 microm to 100 microm) was also established (EC(50)=6.03 microm). During continuous haemorrhage, MAP was maintained in animals injected with 8-Br-cGMP, relative to the control group. Although no change in baseline MAP was observed as a result of intrathecal ODQ injection (10 microl, 100 mM), a greater rate of fall in MAP was observed during haemorrhage. Injecting L-arginine (10, 100, 1000 microm, 10 microl) showed a pressor effect that was consistent with the effect of the downstream messenger, cGMP. Furthermore, its pressor effect was blocked by ODQ pre-administration. The results indicate that cGMP increases blood pressure, and thus suggest that cGMP increases SNA. This supports the hypothesis that the sympathoexcitatory effects of spinal delivery of NO are mediated by a cGMP-dependent mechanism.

The nuclear oncoprotein TLX1/HOX11 associates with pericentromeric satellite 2 DNA in leukemic T-cells.

TLX1/HOX11, a DNA-binding homeodomain protein, was originally identified by virtue of its aberrant expression in T-cell leukemia and subsequently found to be crucial for normal spleen development. The precise mechanism of TLX1 function remains poorly understood, although it is known that it can act as both a transcriptional activator and repressor and can downregulate the Aldh1a1 gene in embryonic mouse spleen. Using a whole-genome PCR approach, we show here that TLX1 protein directly interacts with pericentromeric human satellite 2 DNA sequences. Such DNA is known to localize to heterochromatin, which among other roles has been implicated in gene silencing. The interaction was confirmed in vitro and in vivo by gel retardation and chromatin immunoprecipitation assays involving satellite 2 DNA, which contained sequences resembling TLX1 binding sites. Using immunofluorescence microscopy, TLX1 demonstrated a punctate pattern of staining in the nuclei of leukemic T-cells (ALL-SIL). Double labelling indicated that TLX1 colocalized with the centromeric protein CENP-B, demonstrating that the TLX1 foci corresponded to clusters of centromeric DNA. The novel interaction of TLX1 with constitutive heterochromatin adds an additional level of complexity to the intracellular functions of this transcriptional regulator and may have relevance to its roles in transcriptional repression and T-cell immortalization.

Phosphorylated extracellular signal-regulated kinase 1/2 immunoreactivity identifies a novel subpopulation of sympathetic preganglionic neurons.

Distinct chemical codes are thought to reflect functional specificity in sympathetic preganglionic neurons (SPN). Although a number of chemical candidates have been identified including neurotransmitter-related, calcium-binding and other proteins, signal transduction proteins have been largely neglected. Not only might these chemicals allow discrimination of functionally unique chemical signatures, but they may also identify activated neurons. Immunoreactivity (ir) to phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was differentially located within the thoracic spinal cord depending upon which of three forms of killing was used: the only exception to this was the intermediolateral cell column (IML) which was consistently, densely labeled. The presence or absence of p-ERK1/2 in SPN (n=17,541) within the IML of the thoraco-lumbar spinal cord was determined in seven rats. SPN were identified on the basis of their location, size and that they contained choline acetyltransferase ir. On average, 58% of SPN contained p-ERK1/2, however, more SPN in both the upper (72%; C8-T4) and lower (78%; T11-L3) thoraco-lumbar spinal cord contained p-ERK1/2-ir than the middle thoracic region (47%; T4-T10). p-ERK1/2-ir was also examined in SPN (n=1895) innervating the adrenal medulla (identified by retrograde tracing using cholera toxin B subunit) combined with localization of neuronal nitric oxide synthase (nNOS) in three rats. On average, 64% of adrenal SPN contain p-ERK1/2-ir, and it was confirmed that all adrenal SPN contain nNOS-ir. It appears that p-ERK1/2-ir SPN, described in this study, have tonically activated receptors that are coupled to intracellular signal transduction pathways that lead to the phosphorylation of ERK1/2.

Seasonal variation in preeclampsia based on timing of conception.

OBJECTIVE: Studies have suggested that the incidence of preeclampsia may be partially dependent on the month or season of delivery. We sought to evaluate whether preeclampsia occurs seasonally in our population and whether the timing of conception or delivery is more strongly associated with risk. METHODS: Between January 1995 and August 2003, we identified 142 primiparous women with singleton pregnancies who met the American College of Obstetricians and Gynecologists' definition for preeclampsia and compared them with 7,762 primiparous control deliveries. We analyzed rates of preeclampsia by individual month and 3-month seasonal blocks based on conception and delivery. Data were analyzed with the chi2 test, and logistic regression and odds ratios were calculated where appropriate. RESULTS: Preeclampsia occurred in 1.8% of singleton primiparous gestations (142/7,904). Cases were younger than controls (26.5 +/- 5.6 versus 28.0 +/- 0 6.0 years, P < .003), of similar race (97% white versus 96% white, P = .69), and equally likely to have a female child (45% versus 48%, P = .41). We found no significant association of month (logistic regression P = .20) of delivery with the risk of preeclampsia. There was a significant association of month (P = .003) of conception with risk of preeclampsia. Conception during the summer months had the highest risk (incidence 2.3%; odds ratio 1.7; 95% confidence limits 1.06, 2.75) compared with spring (incidence 1.4%). Fall (1.7%) and winter (1.6%) conceptions were associated with intermediate rates of preeclampsia. CONCLUSION: We identified a seasonal variation in preeclampsia that appears to be more strongly related to timing of conception than to the timing of delivery. The highest incidence of preeclampsia was associated with conception in the summer months. LEVEL OF EVIDENCE: II-2.

Effects of supplementary selenium source on the performance and blood measurements in beef cows and their calves.

On December 2, 1999, 120 pregnant cows were weighed, their body condition scored, and then sorted into six groups of 20 stratified by BCS, BW, breed, and age. Groups were assigned randomly to six, 5.1-ha dormant common bermudagrass (Cynodon dactylon [L.] Pers.) pastures for 2 yr to determine the effects of supplemental Se and its source on performance and blood measurements. During the winter, each group of cows had ad libitum access to bermudagrass/dallisgrass (Paspalum dilatatum Poir.) hay plus they were allowed limited access (1 to 4 d/wk) to a 2.4-ha winter-annual paddock planted in half the pasture. Treatments were assigned randomly to pastures (two pastures per treatment), and cows had ad libitum access to one of three free-choice minerals: 1) no supplemental Se, 2) 26 mg of supplemental Se from sodium selenite/kg, and 3) 26 mg of supplemental Se from seleno-yeast/kg (designed intake = 113 g/cow daily). Data were analyzed using a mixed model; year was the random effect and treatment was the fixed effect. Selenium supplementation or its source had no effect (P > or = 0.19) on cow BW, BCS, conception rate, postpartum interval, or hay DMI. Birth date, birth weight, BW, total BW gain, mortality, and ADG of calves were not affected (P > 0.20) by Se or its source. Whole blood Se concentrations and glutathione peroxidase (GSH-Px) activity at the beginning of the trial did not differ (P > or = 0.17) between cows receiving no Se and cows supplemented with Se or between Se sources. At the beginning of the calving and breeding seasons, cows supplemented with Se had greater (P < 0.01) whole blood Se concentrations and GSH-Px activities than cows receiving no supplemental Se; cows fed selenoyeast had greater (P < or = 0.05) whole blood Se concentrations than cows fed sodium selenite, but GSH-Px did not differ (P > or = 0.60) between the two sources. At birth and on May 24 (near peak lactation), calves from cows supplemented with Se had greater (P < or = 0.06) whole blood Se concentrations than calves from cows fed no Se. At birth, calves from cows fed seleno-yeast had greater (P < or = 0.05) whole blood Se concentrations and GSH-Px activities than calves from cows fed sodium selenite. Although no differences were noted in cow and calf performance, significant increases were noted in whole blood Se concentrations and GSH-Px activities in calves at birth as a result of feeding of seleno-yeast compared to no Se or sodium selenite.

Differential expression of catecholamine synthetic enzymes in the caudal ventral pons.

The analysis of colocalization of multiple catecholamine biosynthetic enzymes within the ventrolateral part of the medulla oblongata of the rat revealed distinct subpopulations of neurons within the C1 region (Phillips et al., J Comp Neurol 2001, 432:20-34). In extending this study to include the caudal pons, it was shown for the first time that the A5 cell group could be distinguished by the presence of immunoreactivity to tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase (AADC), and dopamine beta hydroxylase (DBH). A novel cell group was also identified. The cells within this new group were immunoreactive to DBH but not TH, AADC, or phenylethanolamine N-methyltransferase (PNMT) and will be referred to as the TH-, DBH+ cell group. The TH-, DBH+ neurons were not immunoreactive for either the dopamine or noradrenaline transporters, suggesting that these neurons do not take up these transmitters. A5 neurons were immunoreactive for the noradrenaline transporter but not the dopamine transporter (as previously shown). Retrograde tracing with cholera toxin B revealed that the TH-, DBH+ neurons do not project to the thoracic spinal cord or to the rostral ventrolateral medulla, but A5 neurons do. A calbindin immunoreactive cell group is located in a region overlapping TH-, DBH+ cell group. However, only a few neurons were immunoreactive for both markers. The physiological role of the TH-, DBH+ cell group remains to be determined.

Modulation of ACh-induced currents in rat adrenal chromaffin cells by ligands of alpha2 adrenergic and imidazoline receptors.

The aim of this study was to investigate the expression of the alpha2-adrenergic receptors in the adrenal medulla, and to examine the mechanism by which clonidine and related drugs inhibit acetylcholine (ACh)-induced whole-cell currents in adrenal chromaffin cells. Reverse transcription-polymerase chain reaction (RT-PCR) performed on punches of rat adrenal medulla demonstrated expression of mRNA for the 2A-, alpha2B- and alpha2C-adrenergic receptors. Similar experiments conducted with tissue punches obtained from the adrenal cortex did not reveal expression of these receptor subtypes. Whole-cell currents were recorded in isolated chromaffin cells using the perforated-patch configuration. ACh (50 microM) evoked inward currents with a peak amplitude of 117.8+/-9.3 pA (n = 45; Vhol = -60 mV). The currents were inhibited in a dose-dependent manner (0.5-50 microM) by clonidine, UK 14,304 and rilmenidine (agonists of alpha2/imidazoline receptors), as well as by SKF 86466 and efaroxan (antagonists). Adrenaline and noradrenaline (50-100 microM) had no significant effect. Thus, although the adrenal medulla expresses mRNA for the alpha2-adrenergic receptors, the lack of agonist-antagonist specificity observed in our whole-cell recordings (in the absence of intracellular dialysis) provides additional evidence against the possibility that these inhibitory effects are mediated by classical alpha2 or imidazoline receptor interactions.

Differential expression of catecholamine biosynthetic enzymes in the rat ventrolateral medulla.

Adrenergic (C1) neurons located in the rostral ventrolateral medulla are considered a key component in the control of arterial blood pressure. Classically, C1 cells have been identified by their immunoreactivity for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and/or phenylethanolamine N-methyltransferase (PNMT). However, no studies have simultaneously demonstrated the expression of aromatic L-amino acid decarboxylase (AADC) and dopamine beta-hydroxylase (DBH) in these neurons. We examined the expression and colocalization of all four enzymes in the rat ventrolateral medulla using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Retrograde tracer injected into thoracic spinal segments T2-T4 was used to identify bulbospinal neurons. Using fluorescence and confocal microscopy, most cells of the C1 group were shown to be double or triple labeled with TH, DBH, and PNMT, whereas only 65-78% were immunoreactive for AADC. Cells that lacked detectable immunoreactivity for AADC were located in the rostral C1 region, and approximately 50% were spinally projecting. Some cells in this area lacked DBH immunoreactivity (6.5-8.3%) but were positive for TH and/or PNMT. Small numbers of cells were immunoreactive for only one of the four enzymes. Numerous fibres that were immunoreactive for DBH but not for TH or PNMT were noted in the rostral C1 region. Single-cell RT-PCR analysis conducted on spinally projecting C1 neurons indicated that only 76.5% of cells that contained mRNA for TH, DBH, and PNMT contained detectable message for AADC. These experiments suggest that a proportion of C1 cells may not express all of the enzymes necessary for adrenaline synthesis.

Differential expression of the noradrenaline transporter in adrenergic chromaffin cells, ganglion cells and nerve fibres of the rat adrenal medulla.

Expression of the noradrenaline transporter (NAT) was identified in various cell and fibre populations of the rat adrenal medulla, examined with immunohistochemistry and confocal microscopy. Immunoreactivity for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), aromatic-L-amino-acid decarboxylase (AADC) and dopamine beta-hydroxylase (DBH) was present in all chromaffin cells, while phenylethanolamine N-methyltransferase (PNMT) was used to determine adrenergic chromaffin cell groups. Labelling with NAT antibody was predominantly cytoplasmic and colocalised with PNMT immunoreactivity. Noradrenergic chromaffin cells were not NAT immunoreactive. Additionally, NAT antibody labelling demonstrated clusters of ganglion cells (presumably Type I) and nerve fibres. Expression of TH, AADC, DBH, PNMT and NAT mRNA was examined using reverse transcription-polymerase chain reaction (RT-PCR) from adrenal medulla punches and single chromaffin cells, and results were consistent with those obtained with immunocytochemistry. Chromaffin cells and fibres labelled with antibodies against growth associated protein-43 (GAP-43) were not NAT immunoreactive, while ganglion cells were doubled labelled with the two antibodies. The presence of NAT in adrenergic chromaffin cells, and its absence from noradrenergic cells, suggests that the adrenergic cell type is primarily responsible for uptake of catecholamines in the adrenal medulla.

Heterogeneous control of blood flow amongst different vascular beds.

The control and maintenance of vascular tone is due to a balance between vasoconstrictor and vasodilator pathways. Vasomotor responses to neural, metabolic and physical factors vary between vessels in different vascular beds, as well as along the same bed, particularly as vessels become smaller. These differences result from variation in the composition of neurotransmitters released by perivascular nerves, variation in the array and activation of receptor subtypes expressed in different vascular beds and variation in the signal transduction pathways activated in either the vascular smooth muscle or endothelial cells. As the study of vasomotor responses often requires pre-existing tone, some of the reported heterogeneity in the relative contributions of different vasodilator mechanisms may be compounded by different experimental conditions. Biochemical variations, such as the expression of ion channels, connexin subtypes and other important components of second messenger cascades, have been documented in the smooth muscle and endothelial cells in different parts of the body. Anatomical variations, in the presence and prevalence of gap junctions between smooth muscle cells, between endothelial cells and at myoendothelial gap junctions, between the two cell layers, have also been described. These factors will contribute further to the heterogeneity in local and conducted responses.