Effects of butyrate supplementation on blood glucagon-like peptide-2 concentration and gastrointestinal functions of lactating dairy cows fed diets differing in starch content.

The objective of this study was to evaluate effects of butyrate supplementation on plasma concentration of glucagon-like peptide-2 (GLP-2), apparent total-tract digestibility, and responses to a grain challenge of lactating dairy cows fed diets differing in starch content. Eight Holstein cows averaging 58.6 ± 9.96 d in milk (4 primiparous cows fitted with rumen cannula and 4 multiparous intact cows) were blocked by parity and assigned to one of two 4 × 4 Latin squares balanced for carryover effects with a 2 × 2 factorial arrangement of treatments. Treatments were dietary starch content [20.6 vs. 27.5%, respectively, for low starch (LS) and high starch (HS)] and butyrate supplementation (butyrate vs. control) with 21-d periods. Butyrate was provided as Gustor BP70 WS (Norel, S.A., Madrid, Spain), containing 70% sodium butyrate and 30% fatty acid mixture, at 2% of dietary dry matter (providing butyrate at 1.1% of dietary dry matter), and control premix contained 70% wheat bran and 30% fatty acid mixture. Feeds, orts, and fecal samples were collected from d 17 to 19 to determine apparent total-tract nutrient digestibility. Blood and rumen fluid samples were collected on d 19. The baseline of dry matter intake (DMI) was determined as average DMI from d 17 to 19 for each cow, and cows were feed-restricted at 60% of the baseline DMI on d 20, and a grain challenge was conducted by providing steam-flaked corn grain at 0.6% of body weight, on an as-fed basis, in addition to each treatment diet on d 21, and blood and ruminal fluid samples were collected. The interaction of dietary starch content by butyrate supplementation was significant for plasma GLP-2 concentration, being greater for cows fed butyrate with the HS diet than those fed the other 3 diets. Cows fed butyrate increased n-butyrate concentration in the ruminal fluid and tended to increase dry matter and organic matter digestibility compared with the control. During the grain challenge, rumen endotoxin concentration increased over time and was higher for cows fed the HS diets compared with those fed LS diets. However, response variables related to inflammation were not affected by the grain challenge. However, serum haptoglobin, lipopolysaccharide-binding protein, and serum amyloid-A concentrations were greater for cows fed butyrate with the LS diet, but not for those fed the HS diet. These results indicate that butyrate supplementation may increase plasma GLP-2 concentration for cows fed HS diets, and total-tract digestibility regardless of dietary starch content. However, butyrate supplementation did not mitigate inflammation in this study.

Short communication: Effects of butyrate supplementation on the productivity of lactating dairy cows fed diets differing in starch content.

The objective of this study was to evaluate the effects of butyrate supplementation on the dry matter intake (DMI), milk production, and blood metabolites of lactating dairy cows fed diets differing in starch content. Eight Holstein cows after peak lactation (58.6 ± 9.96 d in milk; mean ± SD) were blocked by parity and assigned to 1 of 2 Latin squares (4 × 4) balanced for carryover effects with a 2 × 2 factorial arrangement of treatments. Treatments differed by dietary starch content (20.6 vs. 27.5%) and butyrate supplementation (butyrate vs. control) with 21-d periods. Experimental diets contained 36 and 30% corn silage, 18 and 15% grass silage, and 46 and 55% concentrates, respectively, for low starch and high starch diets, on a dry matter (DM) basis. Butyrate was provided as Gustor BP70 WS (Norel S.A., Madrid, Spain), containing 70% sodium butyrate and 30% fatty acid mixture, at 2% of dietary DM (providing butyrate at 1.1% of dietary DM), and control premix contained 70% wheat bran and 30% fatty acid mixture. Interaction effects between dietary starch content and butyrate supplementation were not observed for primary response variables, and milk yield was not affected by treatment. Butyrate supplementation increased serum ß-hydroxybutyrate concentration compared with control (0.706 vs. 0.930 mM), but did not exceed 1.2 mM, a commonly accepted value for subclinical ketosis, and DMI was not affected. Cows fed butyrate had increased milk fat content (4.58 vs. 4.37%) and milk fat yield (1.51 vs. 1.42 kg/d), tended to have increased 4% fat-corrected milk yield (35.9 vs. 34.3 kg/d) and feed efficiency (1.56 vs. 1.50; 4% fat-corrected milk yield/DMI), and had decreased milk urea nitrogen (MUN) concentration (10.8 vs. 11.7 mg/dL) compared with control. Cows fed high starch diets tended to have increased DMI (23.3 vs. 22.5 kg/d), increased milk protein yield (1.13 vs. 1.05 kg/d), and decreased MUN concentration (10.3 vs. 12.2 mg/dL). Inclusion of butyrate at 1.1% of dietary DM increased milk fat production and decreased MUN concentration without affecting DMI or increasing the risk of subclinical ketosis, regardless of dietary starch content.

Effect of feeding calf starter with calcium salts of medium-chain fatty acids on the growth and metabolic hormones in calves.

We investigated the effects of a calf starter supplemented with calcium salts of medium-chain fatty acids (MCFA-Ca) on growth and plasma hormone concentration in calves. Twelve Holstein calves were randomly assigned to two dietary groups (without supplementation [CON] and supplemented with MCFA-Ca [MCFA]) from 4 d of age. Calves were fed 1.0 kg/d of milk replacer until 5 wk of age and were completely weaned at 7 wk of age. Calves in the MCFA group received a calf starter containing 1% MCFA-Ca. dry matter intake (DMI) was measured daily, and body weight was measured weekly. Rumen fluid was collected at 13 wk of age to measure pH and volatile fatty acid concentration. Preprandial blood samples were collected weekly to measure the basal plasma hormone and metabolite concentrations. At 4, 8, and 13 wk of age, peri-prandial blood samples were collected every 30 min, from 60 min before feeding to 120 min after feeding, to observe metabolic responses to feeding. In addition, insulin sensitivity was assessed using euglycemic-hyperinsulinemic clamps at 4, 8, and 13 wk of age in three calves from each treatment. There were no differences in starter and hay DMI between the treatments. However, the average daily gain (ADG) after weaning was higher in the MCFA group than in the CON group. Weekly changes in plasma parameters did not differ between the treatments. Plasma concentrations of preprandial ghrelin and postprandial total ketone bodies at 13 wk of age were higher in the MCFA group than in the CON group. At 8 wk of age, peri-prandial plasma insulin concentrations were lower in the MCFA group than in the CON group. There were no differences between the treatments in terms of insulin sensitivity. The present study suggested that feeding weaning calves MCFA-Ca increases the ADG during the postweaning period, which may be mediated by endocrine signals, such as enhanced ghrelin secretion and decreased insulin secretion, without altering insulin sensitivity.

Calves are prone to growth retardation because of insufficient energy intake during the weaning transition period. Starch is the main energy source used in the formulation of calf starters. However, there is a concern that preweaned calves do not have sufficient functional rumen and small intestine to digest large amounts of starch, causing diarrhea, and decreased feed intake. Medium-chain fatty acids are easily accessible to calves and are expected to have functional properties, such as increasing the plasma concentration of ghrelin, which may enhance growth by stimulating growth hormone. The effect of calf starter supplementation with medium-chain fatty acids on growth performance and metabolism has not been evaluated previously and was evaluated in this study. Medium-chain fatty acids were fed in the form of calcium salts as pelleted solid feed. The results showed that feeding medium-chain fatty acids increased plasma ghrelin concentration, decreased insulin concentration, suggesting that these metabolic changes might be beneficial for calf growth performance.

Evaluation of relationship between ruminal pH and the proportion of de novo fatty acids in milk.

The objective of this study was to evaluate the relationship between ruminal pH and milk de novo fatty acid (DNFA) concentrations determined by Fourier-transform infrared spectroscopy. Data were collected from 18 multiparous Holstein cows fitted with a rumen cannula and fed 1 of the experimental diets differing in starch content (22.1 vs. 28.3%) with or without supplementation of a Saccharomyces cerevisiae fermentation product in a previous study. Milk was sampled on d 7 and 21 after calving, and concentrations of milk fat, DNFA (C6 to C14), mixed-origin fatty acids (FA; C16:0 and C16:1), and preformed FA (≥C18) were estimated using Fourier-transform infrared spectrometry. Ruminal pH was recorded in the ventral sac every 30 s continuously for 72 h on d 7 to 9 and 21 to 23 after calving. Daily maximum, nadir, and mean ruminal pH as well as duration and area below pH 5.8 were determined for each period. Milk DNFA (g/100 g of FA) was positively related to nadir (r = 0.428) and mean (r = 0.471) ruminal pH and negatively related to duration (r = -0.511) and area (r = -0.520) below pH 5.8. Milk fat content did not have a relationship with ruminal pH variables in this study. The regression lines for d 7 and 21 were similar, likely because plasma free FA concentrations were not different between d 7 and 21 (513 vs. 534 µEq/L) for the current data set. The coefficients of determination between DNFA and ruminal pH were greater for DNFA in total milk FA (g/100 g of FA) than in milk (g/100 g of milk), suggesting that DNFA in milk fat (g/100 g of FA) is an appropriate measurement variable that relates to ruminal pH even for cows in early lactation.

Effects of fat-enriched diet and methionine on insulin sensitivity in lactating cows.

The hyperinsulinemic-euglycemic clamp (EGC) technique was used to investigate the effects of calcium salts of long-chain fatty acids (LCFA-Ca) and rumen-protected Met (RPM) on insulin sensitivity in the peripheral tissues of lactating cows. Six multiparous Holstein cows were used in a 3 × 3 Latin square experiment in each 14-d period. Dietary treatments were 0 (RPM0), 20 (RPM20), and 60 (RPM60) g/d of RPM, supplemented with a diet containing 1.5% of LCFA-Ca equal to 110% of the cows' ME requirement. And as a control for the 3 LCFA-Ca-containing diets, a dietary treatment without LCFA-Ca (Con) was also included. After a 10-d adaptation period, milk samples were collected for 4 d, and EGC experiments were performed on d 14 of each treatment period. Insulin solution was infused through a jugular vein catheter at a rate of 0.1, 0.2, 0.3, and 0.4 milliunits·kg BW-1·min-1 for 30 min and then at a rate of 0.5 milliunits·kg BW-1·min-1 for 60 min. Glucose solution was variably infused to maintain plasma glucose at steady state through the same catheter. Blood samples for measurements were taken using the contralateral catheter. Plasma total cholesterol, cholesterol ester, free cholesterol, and phospholipid concentrations in RPM0 and RPM20 were higher than those in Con, whereas the concentrations in RPM60 were low at the same degree of those in RPM0 (P < 0.05). Plasma Met concentration was greatest in RPM60 (P < 0.05). In the EGC experiment, the glucose infusion rate was greater in RPM60 than in RPM0 and RPM20 and an effective concentration of insulin resulting in 50% maximal glucose infusion rate was lower in RPM60 compared with RPM0 (P < 0.05), indicating that insulin sensitivity was intensified in RPM60. Although the insulin sensitivity evaluated from the EGC data in RPM0, RPM20, and RPM60 was not different from Con, a slight decline was observed in RPM0 and insulin sensitivity in RPM60 was higher than Con. Our results from the EGC experiment demonstrated that the feeding RPM lead to increased insulin sensitivity, which suggests that dietary Met affects lipid metabolism via insulin action in lactating dairy cows fed a LCFA-Ca-containing diet.

Effect of disulfiram in combination with L-tryptophan and lithium on pentylenetetrazol-induced seizure.

Disulfiram prolonged the latency to clonic seizure caused by pentylenetetrazol (PTZ, 100 mg/kg SC). The effect of disulfiram was augmented by combination with tryptophan plus lithium, although neither tryptophan or lithium prolonged the latency to clonic seizure. The latency to tonic seizure was also prolonged in disulfiram-treated animals in parallel with the prolongation of the latency to clonic seizure. Lithium treatment completely prevented the incidence of tonic seizure, while this effect was cancelled in disulfiram-treated animals. Disulfiram acts on the clonic and tonic seizures in different ways.

Tryptophol-induced change in Brain 5-hydroxytryptamine Metabolism.

The effects of tryptoiphol (TOL) on brain 5-hydroxytryptamine (5-HT) metabolism were studied. After TOL injection (200 mg/kg IP), brain 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels were increased, 5-HT synthesis rate was decreased, and monoamine oxidase (MAO) activity remained unchanged. Pretreatment of mice with p-chlorophenylalanine (PCPA), a potent inhibitor of 5-HT synthesis, did not affect the anticonvulsant action of TOL.. These results suggest that alteration of 5-HT metabolism after TOL injection is not directly related to the anticonvulsant action of TOL

Effect of p-chlorophenylalanine on diazepam withdrawal signs in rats.

The effect of p-chlorophenylalanine (PCPA), a specific serotonin (5-HT) depleter, on diazepam withdrawal signs was studied. Rats were made dependent on diazepam by the chronic administration of this drug in the diet. At the time of diazepam withdrawal, the animals were treated with PCPA (200 mg/kg, IP) or the corresponding vehicle (control). After diazepam withdrawal, the maximal body weight losses of control and PCPA-treated animals were 4.1% and 9.0%, respectively. In naive animals, PCPA did not cause any change in body weight. These results suggest that depletion of central 5-HT by PCPA may potentiate the severity of diazepam withdrawal signs.

Enhanced cholinergic activity in the medulla oblongata of DOCA-salt hypertensive and renal hypertensive rats.

We previously demonstrated that cholinergic activity in the medulla oblongata is enhanced in adult spontaneously hypertensive rats (SHR), a genetically hypertensive rat model. In this study, we examined possible alterations of medulla oblongata cholinergic mechanisms in nongenetic forms of hypertension, using deoxycorticosterone acetate (DOCA)-salt hypertensive and renal hypertensive rats. At a fully developed stage of hypertension in DOCA-salt hypertensive and renal hypertensive rats, choline acetyl-transferase (CAT) activity in the rostro-ventral medulla oblongata was enhanced, whereas there was no change in the activity of CAT in other parts of the medulla oblongata. There was no alteration of the medulla CAT activity in prehypertensive SHR or at an early stage of renal hypertension. Increases in blood pressure and plasma catecholamine levels induced by physostigmine (0.5 mg/kg, i.p.) were enhanced in DOCA-salt hypertensive and renal hypertensive rats. These findings suggest that cholinergic activities in the medulla oblongata are enhanced and that such activities are involved in enhancement of the sympathetic nervous system in non-genetically hypertensive rats. It seems unlikely that the altered cholinergic activity in the rostral ventrolateral medulla of adult SHR occurs genetically.

Evidence suggesting that lateral parabrachial nucleus is responsible for enhanced medullary cholinergic activity in hypertension.

Cholinergic activities in the rostral ventrolateral medulla are enhanced in hypertensive animals, and enhanced cholinergic activity contributes to hypertension. Neurons in the lateral parabrachial nucleus and baroreceptors are suggested to be involved in mediation of acetylcholine release in the rostral ventrolateral medulla. To investigate the hypothesis that the lateral parabrachial nucleus is involved in the increased medullary cholinergic activity in hypertensive rats, we measured choline acetyltransferase activity in the rostral ventrolateral medulla, and examined effects of electrolytic lesioning of the lateral parabrachial nucleus in deoxycorticosterone acetate-salt hypertensive rats. We found that choline acetyl-transferase activity in the medullary region was increased in deoxycorticosterone acetate-salt hypertensive rats. Unilateral lesioning of the lateral parabrachial nucleus abolished the increase in choline acetyltransferase activity in the lesioned side of the medullary region of hypertensive rats, whereas such activity in the medullary region of control rats was unaffected by lesioning. Bilateral lesioning of the lateral parabrachial nucleus inhibited the development of hypertension in hypertensive rats. In contrast, baroreceptor denervation did not inhibit the increase in choline acetyltransferase activity in the medullary region of hypertensive rats. These results are compatible with the hypothesis that the lateral parabrachial nucleus area is involved in mediation of increased medullary cholinergic activity and thus plays a role in the development of hypertension.

Blockade of angiotensin receptors in the anterior hypothalamic preoptic area lowers blood pressure in DOCA-salt hypertensive rats.

It has been established that deoxycorticosterone acetate (DOCA)-salt hypertensive rats have an overactive brain angiotensin-system. The purpose of the present study was to identify the brain sites showing enhanced angiotensin-system activity responsible for the pathogenesis of hypertension in DOCA-salt hypertensive rats. The angiotensin receptor antagonist, losartan, was injected into brain ventricles or into tissues around the rostral parts of the third ventricle in conscious DOCA-salt hypertensive rats. Losartan (1 microg) injection into the lateral ventricle or into the rostral parts of the third ventricle produced a depressor response, whereas the agent did not affect blood pressure when injected into the caudal parts of the third ventricle or into the fourth ventricle. Losartan (0.1 microg) injection into the anterior hypothalamic preoptic area, anterior (AHA) produced a depressor response. Angiotensin II (0.1-1 ng) injection into the AHA produced a pressor response in sham-operated and DOCA-salt hypertensive rats, and the pressor response to angiotensin II (1 ng) was greater in DOCA-salt hypertensive rats than that in sham-operated rats. Release of angiotensin peptides in the AHA was greater in DOCA-salt hypertensive rats than that in sham-operated rats. These findings suggest that the angiotensin-system in the AHA is enhanced, and that this enhancement is involved in the maintenance of hypertension in DOCA-salt hypertensive rats. Both increased pressor reactivity to angiotensin II and increased release of angiotensin peptides in the AHA appear to be related to this enhancement of the angiotensin-system in DOCA-salt hypertensive rats.

Evidence for involvement of the lateral parabrachial nucleus in mediation of cholinergic inputs to neurons in the rostral ventrolateral medulla of the rat.

We examined whether sites in the lateral parabrachial nucleus (PBN) where L-glutamate produced increases in arterial pressure were involved in mediation of cholinergic inputs to neurons in the rostral ventrolateral medulla (RVLM). Male Wistar rats were anesthetized, paralyzed and artificially ventilated. Unilateral microinjection of L-glutamate into the lateral PBN produced a pressor response. Microinjection of the muscarinic receptor antagonist scopolamine into the unilateral RVLM inhibited the pressor response to L-glutamate injected ipsilaterally into the lateral PBN, whereas microinjection of the cholinesterase inhibitor physostigmine into the RVLM enhanced it. PBN microinjection of L-glutamate also enhanced the firing rate of RVLM sympathoexcitatory neurons and the enhancement of the firing rate was inhibited by scopolamine iontophoretically applied on neurons. PBN injection of L-glutamate produced a tetrodotoxin (TTX)-sensitive release of ACh in the RVLM. Unilateral microinjection of TTX into the lateral PBN inhibited the pressor response induced by RVLM microinjection of physostigmine. These results provide evidence that neurons in the pressor sites of the lateral PBN are involved in mediation of cholinergic inputs responsible for pressor responses in the RVLM.

Enhanced release of acetylcholine in the rostral ventrolateral medulla of spontaneously hypertensive rats.

We examined whether the altered rostral ventrolateral medulla (RVLM) cholinergic function in spontaneously hypertensive rats (SHR) results from enhanced presynaptic cholinergic tone. Male 12- to 16-week-old SHR and age-matched Wistar Kyoto rats (WKY) were anesthetized, paralyzed and artificially ventilated. Unilateral microinjection of cholinergic agents into the RVLM produced a pressor response. The pressor response to physostigmine was greater in SHR than that of WKY whereas the response to ACh and carbachol was the same in WKY and SHR. Bilateral microinjection of scopolamine produced a decrease in blood pressure. The depressor response was greater in SHR than that of WKY. When a microdialysis probe was placed in the RVLM, ACh release in the RVLM was greater in SHR than that of WKY. Choline acetyltransferase (CAT) activity was increased only in the rostro-ventral part of the medulla, which contained the RVLM, but not in other parts of the medulla oblongata. Physostigmine (0.5 mg/kg, i.p.)-induced increases in ACh content were also enhanced only in the rostro-ventral part of the medulla. These results provide direct evidence that ACh release in the RVLM is enhanced in SHR. It appears that the enhanced cholinergic activity in the RVLM of SHR results from an increase in cholinergic impulse flow in the RVLM of SHR. This abnormality may play a role in the maintenance of hypertension in SHR.

Angiotensin II mediates pressure loading-induced mitogen-activated protein kinase activation in isolated rat aorta.

Vascular hypertrophy occurs during chronic hypertension and contributes to the elevation of peripheral vascular resistance in hypertension. In this study, we examined whether acute pressure overloading of the vascular wall produces activation of mitogen-activated protein (MAP) kinases, enzymes believed to be involved in the pathway for cell proliferation, in isolated perfused rat aortae, and examined whether the mechanical overloading-induced MAP kinase activation is mediated via the vascular angiotensin system. Aortae were perfused with Tyrode solution. Increases in perfusion pressure caused a pressure-dependent increase in MAP kinase activity in endothelium-intact aortae and in endothelium-denuded aortae. The increase in MAP kinase activity induced by pressure loading was inhibited by the angiotensin receptor antagonist, losartan, the renin inhibitor, pepstatin A, and the angiotensin-converting enzyme inhibitor, captopril. Ca(2+) depletion and the Ca(2+) channel antagonist, nifedipine, did not affect the pressure loading-induced MAP kinase activation. The results of the present study suggest that pressure loading of the vascular wall per se can activate MAP kinases in the vasculature and that the MAP kinase activation is mediated at least partly via the vascular angiotensin system. It seems unlikely that the pressure loading-induced increase in MAP kinase activity is mainly mediated via increases in Ca(2+) influx in vascular cells.

Mitogen-activated protein kinase activity regulation role of angiotensin and endothelin systems in vascular smooth muscle cells.

To examine whether angiotensin II and endothelins produced in vascular smooth muscle cells can play roles in the regulation of mitogen-activated protein (MAP) kinase activity in vascular smooth muscle cells, we measured the activity of MAP kinases in cultured vascular smooth muscle cells, and determined effects of renin-angiotensin and endothelin systems activators and inhibitors. Angiotensin II and endothelin-1 produced an activation of MAP kinase activity in vascular smooth muscle cells, whereas the angiotensin receptor antagonist, losartan and the endothelin receptor antagonist, cyclo (D-alpha-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl, BQ123) inhibited the enzyme activity. MAP kinase activity in vascular smooth muscle cells was also inhibited either by the renin inhibitor pepstatin A or by the angiotensin-converting enzyme inhibitor captopril. The degree of the inhibition of MAP kinase activity by pepstatin A, captopril and losartan was almost the same. Renin produced a considerable increase in MAP kinase activity and the renin-induced MAP kinase activation was inhibited by pepstatin A. The endothelin precursor big endothelin-1 produced an increase of MAP kinase activity in vascular smooth muscle cells, whereas the endothelin-converting enzyme inhibitor phosphoramidon inhibited the enzyme activity. These findings suggest that functional renin-angiotensin system and endothelin system are present in vascular smooth muscle cells and these systems tonically serve to increase MAP kinase activity. It appears that renin or renin-like substances play the determining role in the regulation of renin-angiotensin system in vascular smooth muscle cells.

Changes in the serotonin and 5-hydroxyindoleacetic acid contents in rat brain after ethanol and disulfiram treatments.

Disulfiram (700 mg/kg, p.o.) significantly increased the brain levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in rats. Ethanol (1 g/kg and 2 x 1.5 g/kg, i.p.) did not affect the 5-HT level, but at the higher dose it significantly increased the 5-HIAA level. When disulfiram was given in combination with ethanol, the brain level of 5-HT was significantly higher and, conversely, the 5-HIAA level was significantly lower, as compared to the results with disulfiram alone. These results suggest that the biogenic aldehyde derived from 5-HT may influence 5-HT metabolism and the elimination of 5-HIAA from the brain.

Local renin-angiotensin system and mitogen-activated protein kinase activation in rat aorta.

We previously reported that endogenous angiotensin II is released to cause mitogen-activated protein (MAP) kinase stimulation in the media portion of the vasculature. In this study, we examined whether a functional renin-angiotensin system is indeed present within the media of the vasculature. In rat aortic strips, endothelium removal produced an increase of MAP kinase activity. The MAP kinase activation was inhibited either by the renin inhibitor pepstatin A or by the angiotensin-converting enzyme inhibitor captopril. The degree of the inhibition of the MAP kinase activation by pepstatin A, captopril and the angiotensin receptor antagonist losartan was almost the same. Pepstatin A inhibited MAP kinase activation induced by renin but not by angiotensin I and angiotensin II. Captopril inhibited the MAP kinase activation induced by angiotensin I but not by angiotensin II. In nephrectomized rat aortic strips, endothelium removal also produced an increase in MAP kinase activity, but the MAP kinase activation was considerably small and minimally inhibited by losartan. Nephrectomy produced a marked decrease in plasma renin activity. These findings suggest that an apparently fully intact and functional renin-angiotensin system is present in the media of the rat vasculature and this system serves to increase MAP kinase activity. It appears that renin plays the determining role in the regulation of angiotensin generation also in the media and the major source of the renin is renin of kidney origin.

Evidence for the involvement of endogenous aspartate in the mediation of carotid chemoreceptor reflexes in the rostral ventrolateral medulla of the rat.

2-Amino-5-phosphonovalerate (AP5; 153 pmol) injected into the rostral ventrolateral medulla (RVLM) inhibited pressor responses induced by carotid chemoreceptor stimulation. AP5 also inhibited pressor responses to aspartate (0.75 nmol) but not to glutamate (0.53 nmol) similarly injected. High K+ (50 mM) released endogenous aspartate and glutamate in a Ca2+-dependent manner from the RVLM. Chemoreceptor stimulation caused a release of aspartate but not of glutamate in the RVLM, and sinus nerve denervation abolished the release of aspartate. Increases in blood pressure induced by intravenous phenylephrine did not release aspartate. These results support the hypothesis that endogenous aspartate in the rat RVLM is involved in the mediation of chemoreceptor reflexes.

Altered cholinergic mechanisms and blood pressure regulation in the rostral ventrolateral medulla of DOCA-salt hypertensive rats.

We examined whether cholinergic transmission in the rostral ventrolateral medulla (RVLM) of deoxycorticosterone acetate-salt hypertensive rats (DHR) is enhanced and the enhancement is involved in the maintenance of hypertension in DHR, and whether cholineacetyltransferase (ChAT) activities and ChAT mRNA expression are enhanced in neurons intrinsic to the RVLM of DHR. Rats were anesthetized, paralyzed, and artificially ventilated. Unilateral microinjection of cholinergic agents into the RVLM produced a pressor response. The pressor response to physostigmine was greater in DHR than in control rats, whereas the response to carbachol was the same in both sets of rats. Bilateral microinjection of scopolamine into the RVLM produced a decrease in blood pressure. The depressor response was greater in DHR than in control rats. The number of ChAT-activity-detected neurons in the RVLM was greater in DHR than in control rats. The number of ChAT mRNA-expressing neurons in the RVLM was also clearly greater in DHR than in control rats. These results demonstrate that cholinergic transmission in the RVLM is enhanced in DHR, and this enhancement may play a role in the maintenance of hypertension in DHR. It is probable that enhanced activity of cholinergic neurons intrinsic to the RVLM is at least in part, responsible for the enhanced cholinergic transmission in the RVLM of DHR.

Midbrain central gray is involved in mediation of cholinergic inputs to the rostral ventrolateral medulla of the rat.

There are cholinergic inputs responsible for pressor responses in the rostral ventrolateral medulla (RVLM) and stimulation of midbrain central gray (CG) increases arterial pressure via activation of neurons in the RVLM. In this study, we examined whether the CG was involved in mediation of the cholinergic inputs to the RVLM. Male Wistar rats were anesthetized, paralyzed, and artificially ventilated. Unilateral microinjection of L-glutamate into the CG produced a pressor response. Microinjection of the muscarinic receptor antagonist scopolamine into the unilateral RVLM inhibited the pressor response to L-glutamate injected ipsilaterally into the CG, whereas microinjection of the cholinesterase inhibitor physostigmine into the RVLM enhanced it. CG stimulation also enhanced the firing rate of RVLM barosensitive neurons and the enhancement of the firing rate was inhibited by scopolamine iontophoretically applied on neurons. CG injection of L-glutamate produced a release of acetylcholine in the RVLM. Unilateral microinjection of L-glutamate into the pedunculopontine tegmental nucleus (PPT) also produced a pressor response, but the pressor response to L-glutamate was not affected by scopolamine injected ipsilaterally into the RVLM. These results provide evidence that the CG but not the PPT is involved in mediation of cholinergic inputs responsible for pressor responses in the RVLM.