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Long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) may be more bioavailable from krill oil compared to fish oil due to their phospholipid structure. We tested whether a microencapsulated krill and tuna oil blend (ME-TOKO) provided greater LC n-3 PUFA bioavailability, improved blood lipid profiles and increased intestinal contractility compared to microencapsulated tuna oil (ME-TO). Rats were divided into three groups to receive isocaloric diets containing ME-TO, ME-TOKO and microencapsulated olive oil (ME-OO) at 0.3 or 2 g/100 g for 4 weeks. Final body and organ weights, feed intake and waste output were similar. ME-TOKO rats had higher plasma total LC n-3 PUFA levels compared to ME-TO, but liver LC n-3 PUFA levels and plasma triglyceride and cholesterol levels were similar in non-fasted rats. Diets containing 2% ME-TO and ME-TOKO also showed similar increases in ileal contractility. In summary, ME-TO bioavailability of LC n-3 PUFA was similar to ME-TOKO.

Hypertension is an inflammatory condition controlled by the renin angiotensin system and is linked to kidney disease, diabetes mellitus, and recently to dysfunction of the gut. The aim of this study was to determine what effect antihypertensive drug treatments may have on intestinal function of the spontaneously hypertensive rat (SHR). In the first experiment, SHRs were treated with enalapril, hydralazine, or with no treatment as a control. In the second experiment, SHRs were treated with losartan or with no treatment as a control. All drug treatments led to significant lowering of blood pressure after 16 weeks. At termination, intact tissue sections of the ileum and colon were induced to contract ex vivo by KCl; electrical stimulation; and agonists carbachol, angiotensin II, and prostaglandin E 2 (PGE 2 ). There were no differences in ileal or colonic contractility due to hydralazine or enalapril compared with no-treatment SHR control. However, for the ileum, the losartan group responded significantly more to KCl and carbachol while responding less to angiotensin II, with no difference for PGE 2 compared with the no-treatment SHR control. In contrast, the colon responded similarly to KCl, electrical stimulation, and PGE 2 but responded significantly less to angiotensin II. These results demonstrate that the ileum responds differently (with KCl and carbachol as agonists) to the colon after losartan treatment, whereas there is a reduced contractile response in both the ileum and colon following losartan treatment. Although there are few well documented major contraindications for angiotensin receptor blockers, the modulation of gut contractility by losartan may have wider implications for bowel health., (Copyright © 2017 by The Author(s).)

Hypertension is a major risk factor for coronary heart disease, kidney disease, and stroke. Interest in medicinal or nutraceutical plant bioactives to reduce hypertension has increased dramatically. The main biological regulation of mammalian blood pressure is via the renin-angiotensin-aldosterone system. The key enzyme is angiotensin converting enzyme (ACE) that converts angiotensin I into the powerful vasoconstrictor, angiotensin II. Angiotensin II binds to its receptors (AT1) on smooth muscle cells of the arteriole vasculature causing vasoconstriction and elevation of blood pressure. This review focuses on the in vitro and in vivo reports of plant-derived extracts that inhibit ACE activity, block angiotensin II receptor binding and demonstrate hypotensive activity in animal or human studies. We describe 74 families of plants that exhibited significant ACE inhibitory activity and 16 plant families with potential AT1 receptor blocking activity, according to in vitro studies. From 43 plant families including some of those with in vitro bioactivity, the extracts from 73 plant species lowered blood pressure in various normotensive or hypertensive in vivo models by the oral route. Of these, 19 species from 15 families lowered human BP when administered orally. Some of the active plant extracts, isolated bioactives and BP-lowering mechanisms are discussed.

Background and Aim: Dietary fiber shortens gut transit time, but data on the effects of fiber components (including resistant starch, RS) on intestinal contractility are limited. We have examined RS effects in male Sprague-Dawley rats fed either a high-amylose maize starch (HAMS) or a wholemeal made from high-amylose wheat (HAW) on ileal and colonic contractility ex vivo and expression of genes associated with smooth muscle contractility., Methods: Rats were fed diets containing 19 % fat, 20 % protein, and either low-amylose maize starch (LAMS), HAMS, wholemeal low-amylose wheat (LAW) or HAW for 11 week. Isolated ileal and proximal colonic sections were induced to contract electrically, or by receptor-independent (KCl) or receptor-dependent agents. Colonic gene expression was assessed using an Affymetrix microarray., Results: Ileal contractility was unaffected by treatment. Maximal proximal colonic contractility induced electrically or by angiotensin II or carbachol was lower for rats fed HAMS and LAW relative to those fed LAMS (P < 0.05). The colonic expression of genes, including cholinergic receptors (Chrm2, Chrm3), serotonin receptors (Htr5a, Htr7), a protease-activated receptor (F2r), a prokineticin receptor (Prokr1), prokineticin (Prok1), and nitric oxide synthase 2 (Nos2), was altered by dietary HAMS relative to LAMS (P < 0.05). HAW did not significantly affect these genes or colonic contractility relative to effects of LAMS., Conclusions: RS and other fiber components could influence colorectal health through modulation of stool transit time via effects on muscular contractility.

Fish oil n-3 fatty acids (FA) have known health benefits. Microencapsulation stabilises and protects fish oil from oxidation, enabling its incorporation into foods. The aim of the present study was to compare the bioavailability of n-3 FA delivered as two microencapsulated fish oil-formulated powders or fish oil gel capsules (FOGC) taken with a flavoured milk in healthy participants. Formulation 1 (F1) composed of a heated mixture of milk protein-sugar as an encapsulant, and formulation 2 (F2) comprised a heated mixture of milk protein-sugar-resistant starch as an encapsulant. Participants consumed 4 g fish oil (approximately 1·0 g EPA and DHA equivalent per dose). Bioavailability was assessed acutely after ingestion of a single dose by measuring total plasma FA composition over a period of 48 h (n 14) using a randomised cross-over design, and over the short term for a period of 4 weeks using an unblinded parallel design (after daily supplementation) by measuring total plasma and erythrocyte FA composition at baseline and at 2 and 4 weeks (n 47). In the acute study, F1 greatly increased (% Δ) plasma EPA and total n-3 FA levels at 2 and 4 h and DHA levels at 4 h compared with FOGC. The time to reach maximal plasma values (T(max)) was shorter for F1 than for FOGC or F2. In the short-term study, increases in plasma and erythrocyte n-3 FA values were similar for all treatments and achieved an omega-3 index in the range of 5·8-6·3 % after 4 weeks. Overall, the results demonstrated human bioequivalence for microencapsulated fish oil powder compared with FOGC.

Cardiovascular disease is the leading cause of mortality in many economically developed nations, and its incidence is increasing at a rapid rate in emerging economies. Diet and lifestyle issues are closely associated with a myriad of cardiovascular disease risk factors including abnormal plasma lipids, hypertension, insulin resistance, diabetes and obesity, suggesting that diet-based approaches may be of benefit. Omega-3 longchain-polyunsaturated fatty acids (ω3 LC-PUFA) are increasingly being used in the prevention and management of several cardiovascular risk factors. Both the ω3 and ω6 PUFA families are considered essential, as the human body is itself unable to synthesize them. The conversion of the two precursor fatty acids - linoleic acid (18:2ω6) and α-linoleic acid (α18:3ω3) - of these two pathways to longer (≥C(20)) PUFA is inefficient. Although there is an abundance of ω6 PUFA in the food supply; in many populations the relative intake of ω3 LC-PUFA is low with health authorities advocating increased consumption. Fish oil, rich in eicosapentaenoic (EPA, 20:5ω3) and docosahexaenoic (DHA, 22:6ω3) acids, has been found to cause a modest reduction in blood pressure at a dose level of >3g/d both in untreated and treated hypertensives. Whilst a multitude of mechanisms may contribute to the blood pressure lowering action of ω3 LC-PUFA, improved vascular endothelial cell function appears to play a central role. Recent studies which evaluated the potential benefits of fish oil in type-2 diabetes have helped to alleviate concerns raised in some previous studies which used relatively large dose (5-8 g/d) and reported a worsening of glycemic control. Several meta-analyses have confirmed that the most consistent action of ω3 LC-PUFA in insulin resistance and type-2 diabetes is the reduction in triglycerides. In some studies, fish oil has been found to cause a small rise in LDL-cholesterol, but a change in the LDL particle size, from the smaller more atherogenic form to the larger, less damaging particle size, have also been noted. ω3 LC-PUFA are effective modulators of the inflammation that accompanies several cardio-metabolic abnormalities. Taking into consideration the pleiotropic nature of their actions, it can be concluded that dietary supplementation with ω3 LC-PUFA will lead to improvements in cardio-metabolic health parameters. These fatty acids pose only minor side effects and more importantly, do not interact adversely with the common drug therapies used in the management and treatment of hypertension, dyslipidemia, type-2 diabetes, and obesity/metabolic syndrome, but in some instances work synergistically, thereby providing additional cardiovascular benefits.

Background and Aims: Chronic opioid analgesia has the debilitating side-effect of constipation in human patients. The major aims of this study were to: 1) characterize the opioid-specific antagonism of morphine-induced inhibition of electrically driven contraction of the small intestine of mice, rats, and guinea pigs; and 2) test if the oral delivery of small milk-derived opioid antagonist peptides could block morphine-induced inhibition of intestinal transit in mice., Methods: Mouse, rat, and guinea pig intact ileal sections were electrically stimulated to contract and inhibited with morphine in vitro. Morphine inhibition was then blocked by opioid subtype antagonists in the mouse and guinea pig. Using a polymeric dye, Poly R-478, the opioid antagonists casoxin 4 and lactoferroxin A were tested orally for blocking activity of morphine inhibition of gut transit in vivo by single or double gavage techniques., Results: The guinea pig tissue was more sensitive to morphine inhibition compared with the mouse or the rat (IC(50) [half maximal inhibitory concentration] values as nmol/L ± SEM were 34 ± 3, 230 ± 13, and 310 ± 14 respectively) (P < 0.01). The inhibitory influence of opioid agonists (IC(50)) in electrically driven ileal mouse preparations were DADLE ([D-Ala(2), D-Leu(5)]-enkephalin) ≥ met-enkephalin ≥ dynorphin A ≥ DAMGO ([D-Ala(2), N-Me-Phe(4), Glyol(5)]-enkephalin) > morphine > morphiceptin as nmol/L 13.9, 17.3, 19.5, 23.3, 230, and 403 respectively. The mouse demonstrated predominantly κ- and δ-opioid receptor activity with a smaller μ-opioid receptor component. Both mouse and guinea pig tissue were sensitive to casoxin 4 antagonism of morphine inhibition of contraction. In contrast to naloxone, relatively high oral doses of the μ-opioid receptor antagonists, casoxin 4 and lactoferroxin A, applied before and after morphine injection were unable to antagonize morphine inhibition of gut transit., Conclusions: Casoxin 4 reverses morphine-induced inhibition of contraction in mice and guinea pigs in vitro but fails to influence morphine inhibition of mouse small intestinal transit by the oral route.

The short-chain fatty acids acetate, propionate, and butyrate are produced by colonic bacterial fermentation of carbohydrates. Butyrate is important in the regulation of the colonocyte cell cycle and gut motility and may also reduce the risk of large bowel cancer. We have shown that dietary butyrylated starch can deliver butyrate to the large bowel in a sustained manner. We hypothesized that ingestion of butyrylated starch increases large bowel butyrate levels and decreases colonic contractility. Groups of male Sprague-Dawley rats (n = 8) were fed AIN-93G-based diet containing a highly digestible low-amylose maize starch (LAMS) control or 5% or 10% butyrylated LAMS (LAMSB) for 10 days. We found that cecal but not colonic tissue weight as well as cecal and distal colonic digesta weights and fecal output were higher in LAMSB fed rats. Butyrylated LAMS lowered digesta pH throughout the large bowel. Cecal, proximal, and distal colonic butyrate pools and portal venous butyrate concentrations were higher in rats fed LAMSB. Electrically stimulated and receptor-dependent carbachol and prostaglandin E(2)-induced isotonic contractions were lower in isolated intact sections of proximal colon (P < .05) but not the terminal ileum after 10% LAMSB ingestion. These results demonstrated that elevation of butyrate levels in the large bowel of the rat correlated with reduction of contractile activity of the colonic musculature, which may assist in the reabsorption of water and minerals., (Copyright 2010 Elsevier Inc. All rights reserved.)

The aim of this study was to design food grade matrices to deliver microencapsulated fish oil to the large bowel of the rat where the potential exists to retard inflammation and cancer development. Digestion in simulated gastric fluid and intestinal fluid demonstrated that only 4-6% of oil was released from the following dried emulsion formulations: 50% fish oil encapsulated in heated casein-glucose-dried glucose syrup (1:1:1) (Cas-Glu-DGS-50); 25% fish oil in casein-modified resistant starch (Hylon VII) (1:1) (Cas-Hylon-25); or 25% fish oil in Cas-Glu-Hylon (1:1:1) (Cas-Glu-Hylon-25). A short-term gavage study (0-12 h) with fish oil and Cas-Glu-DGS-50 demonstrated the appearance of fish oil long chain (LC) n-3 polyunsaturated fatty acids (PUFA) into the plasma indicating specific small intestinal absorption with little LC n-3 PUFA reaching the large bowel. In a 2-week-long term, daily gavage study, the bioavailability of fish oil and fish oil in Cas-Glu-DGS-50 or Cas-Hylon-25 demonstrated that fish oil and Cas-Glu-DGS-50 LC n-3 PUFA were incorporated into the tissue of the small intestine and colon, whereas Cas-Hylon-25 was resistant to degradation in the small intestine. The use of modified Hylon VII for targeted colonic delivery was confirmed in the final short-term gavage study (0-14 h) using Cas-Glu-Hylon-25 with [(14)C]-trilinolenin as a marker incorporated into the microcapsules, where up to 60% of the labeled oil reached the large bowel. Depending on the microencapsulating matrix employed, fish oil can be delivered selectively to the small intestine or to a high degree to the large bowel.

Angiotensin II (AngII) is an octapeptide hormone with a key role in blood pressure regulation. AngII increases blood pressure by stimulating G protein-coupled receptors in vascular smooth muscle. AngII receptors are therefore an important target in patients with high blood pressure. Strategies to lower high blood pressure (hypertension) include the use of drugs that compete for AngII at the angiotensin II Type 1 receptors (ATR) using ATR antagonists (e.g., irbesartan, valsartan, and losartan). This chapter will demonstrate the subtype specificity of ATR binding and we discuss some of the key experiments that are necessary in optimizing some of the parameters for GPCR screening. The latter protocols include saturation binding to determine K (d) and B (max), as well as competition/inhibition experiments to determine the IC(50) of binding. For these experiments we have used rat liver membranes which express ATR (type 1a) in relatively abundant amounts. Additionally, rat liver membrane preparations can be easily prepared in "bulk," frozen away for extended periods (up to 1 year) and used when necessary with no loss of receptor binding activity using the radiolabeled angiotensin II analogue, [(125)I][Sar(1),I le(8)]AngII.

We have shown independently that dietary fiber and n-3 fatty acids can affect gut function. This study investigated the interactive effects of resistant starch (RS) (as high amylose maize starch [HAMS]) and tuna fish oil on ileal contractility. Four-week-old male Sprague Dawley rats were fed 4 diets that contained 100 g/kg fat as sunflower oil or tuna fish oil, with 10% fiber supplied as alpha -cellulose or HAMS for 6 weeks. Fish oil feeding led to higher ileal n-3 fatty acid levels (mainly as DHA) and higher agonist-induced maximal contractility with an RS effect noted for carbachol. HAMS-containing diets resulted in lower colonic pH and higher total short-chain fatty acids (SCFA), but not for butyrate with fish oil. Low prostanoid responses in young rats were enhanced by fish oil independent of RS. The order of muscarinic receptor subtype responses were different compared to older rats; fish oil feeding altered the sensitivity of the M(1) receptor subtype. Although little interactive effects were demonstrated, these data suggest developmental changes in ileal receptor systems with independent effects of RS and fish oil on some bowel properties in juvenile rats.

We have reported that dietary fish oil (FO) leads to the incorporation of long-chain n-3 PUFA into the gut tissue of small animal models, affecting contractility, particularly of rat ileum. This study examined the FO dose response for the incorporation of n-3 PUFA into ileal tissue and how this correlated with in vitro contractility. Groups of ten to twelve 13-wk-old Wistar-Kyoto rats were fed 0, 1, 2.5, and 5% FO-supplemented diets balanced with sunflower seed oil for 4 wk, after which ileal total phospholipid FA were determined and in vitro contractility assessed. For the total phospholipid fraction, increasing the dietary FO levels led to a significant increase first evident at 1% FO, with a stepwise, nonsaturating six-fold increase in n-3 PUFA as EPA (20:5n-3), DPA (docosapentaenoic acid, 22:5n-3), and DHA, but mainly as DHA (22:6n-3), replacing the n-6 PUFA linoleic acid (18:2n-6) and arachidonic acid (20:4n-6) over the dosage range. There was no difference in KCl-induced depolarization-driven contractility. However, a significant increase in receptor-dependent maximal contractility occurred at 1% FO for carbachol and at 2.5% FO for prostaglandin E2, with a concomitant increase in sensitivity for prostaglandin E2 at 2.5 and 5% FO. These results demonstrate that significant increases in ileal membrane n-3 PUFA occurred at relatively low doses of dietary FO, with differential receptor-dependent increases in contractility observed for muscarinic and prostanoid agonists.

A high plasma cholesterol level, especially low-density lipoprotein cholesterol, indicates increased risk of cardiovascular diseases. Plasma cholesterol levels are influenced by diet and cholesterol biosynthesis, uptake, and secretion. Cholesterol uptake involves solubilization into complex phospholipid spherical bodies termed micelles that facilitate the transport of lipids through the gut brush border membrane into enterocytes. In vitro assays reported to date to determine potential cholesterol-lowering effects of various compounds require artificial micelle preparations that are elaborate and time-consuming to prepare. The aims of this study were to compare the efficacy of artificially prepared micelles with naturally derived micelles from pig's bile and to test their ability to assess potential inhibitors of cholesterol uptake. The suitability of pig's bile-derived micelles was tested both at the level of the micelle and at cellular uptake using cultured Caco-2 cells. Known cholesterol uptake inhibitors at the micelle (green tea catechins) and at the Caco-2 cell (beta-lactoglobulin-derived peptide, IIAEK) were used as reference inhibitory compounds. It was concluded that pig's bile was a rapid, reproducible, convenient, and cost-effective source of micelles for cholesterol micelle solubility and cellular uptake assay systems and is suitable for screening purposes focused on identifying potential cholesterol-lowering agents.

We have reported that dietary fish oil (FO) rich in n-3 PUFA modulates gut contractility. It was further demonstrated that the gut of spontaneously hypertensive rats (SHR) has a depressed contractility response to prostaglandins (PG) compared with normotensive Wistar-Kyoto (WKY) rats. We investigated whether feeding diets supplemented with n-3 PUFA increased gut contractility and restored the depressed prostanoid response in SHR gut. Thirteen-week-old SHR were fed diets containing fat at 5 g/100 g as coconut oil (CO), lard, canola oil containing 10% (w/w) n-3 FA as alpha-linolenic acid (1 8:3n-3), or FO (as HiDHA, 22:6n-3) for 12 wk. A control WKY group was fed 5 g/100 g CO in the diet. As confirmed, the SHR CO group had a significantly lower gut response to PGE2 and PGF2alpha compared with the WKY CO group. Feeding FO increased the maximal contraction response to acetylcholine in the ileum compared with all diets and in the colon compared with lard, and restored the depressed response to PGE2 and PGF2alpha in the ileum but not the colon of SHR. FO feeding also led to a significant increase in gut total phospholipid n-3 PUFA as DHA (22:6n-3) with lower n-6 PUFA as arachidonic acid (20:4n-6). Canola feeding led to a small increase in ileal EPA (20:5n-3) and DHA and in colonic DHA without affecting contractility. However, there was no change in ileal membrane muscarinic binding properties due to FO feeding. This report confirms that dietary FO increases muscarinic- and eicosanoid receptor-induced contractility in ileum and that the depressed prostanoid response in SHR ileum, but not colon, is restored by tissue incorporation of DHA as the active nutrient.

Dietary saturated fat (SF) has adverse effects on cardiac and vascular smooth muscle (VSM) contractility. Furthermore, VSM of spontaneously hypertensive rats (SHR) is overreactive to various biological stimuli. The aim of this study was to investigate the effects of increasing dietary fat as lard on gut contractility in SHR. Control Wistar-Kyoto (WKY) rats and SHR (13 wk old) were fed for 12 wk a diet containing 3% sunflower oil [low fat (LF), 3% total fat] or diets supplemented with 7% lard [medium fat (MF), 10% total fat] or 27% lard [high fat (HF), 30% total fat]. For ileal and colonic tissues (WKY and SHR), there was a lower total phospholipid PUFA (n-6)/(n-3) ratio with increased dietary SF (P < 0.003). For WKY, increasing SF led to lower levels of the major SCFA and lower total SCFA levels in cecal digesta (P < 0.01). This trend was evident in SHR but significant only for butyrate (P < 0.01). Contractility responses were unaltered in ileum. In colon, there was a change of sensitivity (50% effective concentration) to angiotensin II in WKY (P < 0.05) due to increased SF and a change of sensitivity to prostaglandin (PG)E(2) and carbachol in SHR (P < 0.05). When the 3 dietary groups were combined, there was lower sensitivity (P < 0.01) and lower maximal contraction (P < 0.05) in ileum and lower maximal contraction in colon of SHR in response to PGF(2alpha) (P < 0.05) and PGE(2) (P < 0.01) compared with WKY. Unlike (n-3) PUFA, dietary SF had little overall effect on gut contractility. However, this is the first report of a defect in PG responsiveness from gut tissue from hypertensive rats.

The aim of the study was to feed convenience baby food brown rice (BC) and white rice (WC) congee diets compared to egg custard (EC) and baked bean (BB) diets to newborn guinea pig pups. Diets were isocaloric and formulated to contain equal macronutrient content of carbohydrate, protein, fat and fibre. Diets were supplemented with essential nutrients, fruit and vegetables and decrementally with standard chow for palatability. We investigated the acceptability of the diets and specifically whether the different natural fibre content of these diets could influence whole animal and small intestinal growth, caecal digesta properties and specifically in vitro ileal contractility. After 8 weeks of feeding, the mean body weight of WC group was significantly lower than the BB group. WC group had lower small intestine weight than both BC group and BB group resulting in lower small intestine density compared to BB group. Caecal digesta pH and total short chain fatty acid (SCFA) concentration were similar. However, butyrate was higher in the BB group compared to the other diets. Contractility studies revealed a small but significantly higher voltage was required to initiate ileal contraction of BC group compared to both the EC and BB groups. All dietary groups responded similarly to acetylcholine, histamine, serotonin, PGE(2), PGF(2alpha), and 8-iso-PGE(2). There were no differences on inhibition of electrically-driven contraction by morphine or epinephrine. The newborn guinea pig model was an effective system for testing, with limitations, supplemented convenience baby foods with variable natural fibre content that demonstrated significant effects on animal growth, caecal digesta SCFA and intestinal contractility.

The long-chain (n-3) polyunsaturated fatty acids (PUFA) have been reported to exhibit health benefits and healing properties for the gastrointestinal tract. The aim of this study was to investigate the effects of dietary fish oil supplementation on the in vitro contractility of gut tissue. Rats (9 wk old) were fed synthetic diets supplemented with 170 g/kg Sunola oil (SO; 850 g/kg as oleic acid [18:1(n-9)]) or with 100 g/kg of the SO replaced by saturated animal fat (SF) or fish oil (FO) for 4 wk. In the colon, there was no difference in the sensitivity (50% effective concentration) or the maximal contraction among the three dietary groups induced by acetylcholine or 8-iso-prostaglandin (PG)E(2) with the rat colon being relatively insensitive to the thromboxane mimetic U-46619. However, in the ileum, the FO group had greater maximal contractions induced by acetylcholine and 8-iso-PGE(2) compared with the SO and SF groups (P < 0.05), and greater maximal contractions induced by PGE(2), PGF(2alpha) and U-46619 compared with the SF group (P < 0.05). FO feeding increased the incorporation of (n-3) PUFA (eicosapentaenoic [20:5(n-3)], docosapentaenoic [22:5(n-3)] and docosahexaenoic acids [22:6(n-3) primarily at the expense of (n-6) PUFA (linoleic [18:2(n-6)] and arachidonic acids [20:4(n-6)]) in the ileum and colon phospholipid fatty acids (P < 0.05). The FO group had a lower cecal digesta pH (P < 0.001) and a greater butyrate concentration than the SF group (P < 0.05). These results suggest that dietary (n-3) PUFA may modulate the contractility of the small intestine.

A modified apparatus is described that provides for the simultaneous bathing of the serosa of an intact piece of isolated guinea pig ileum while allowing infusion of the isolated lumen. The comparative compartmental potency of the opioid agonists morphine, casomorphins, and enkephalins to inhibit electrically driven contractions are described in this system. The rank-order potency for serosally applied opioid agonists was (IC(50) values, nM): [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin (DAMGO) (15)>[D-Ala(2),D-Leu(5)]-enkephalin (DADLE) (35)> or =morphine (46)> or =[D-Ala(2)]-met-enkephalinamide (55)>[D-Ala(2)]-beta-casomorphin[1--4] amide (122)>beta-casomorphin[1--4] amide (940)>met- and leu-enkephalin (>6000). This contrasted to the rank-order potency for the luminally applied opioid agonists: DADLE (63)>DAMGO (135)>[D-Ala(2)]-met-enkephalinamide=morphine (4700)>[D-Ala(2)]-beta-casomorphin[1--4] amide (29000). beta-Casomorphin[1--4] amide, leu-enkephalin and met-enkephalin are mostly inactive when applied luminally. Furthermore, the opioid antagonists, casoxin 4 and [D-Ala(2)]-casoxin 4, when infused into the lumen, significantly overcame the inhibitory effect of morphine added to the serosal side. This model provides an assay and screening system to differentiate between the effects of chemical agents applied via the blood stream (serosa) or food side (lumen) on quiescent or electrically driven gut activity of the nervous plexi or receptor systems of the ileum.

A protective effect of the n-3 polyunsaturated fatty acids (PUFAs) in preventing ventricular fibrillation in experimental animals and cultured cardiomyocytes has been demonstrated in a number of studies. In this study, a possible role for the n-3 PUFAs in the treatment of atrial fibrillation (AF) was investigated at the cellular level using atrial myocytes isolated from young adult rats as the experimental model. Electrically-stimulated, synchronously-contracting myocytes were induced to contract asynchronously by the addition of 10 microM isoproterenol. Asynchronous contractile activity was reduced following acute addition of the n-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) at 10 microM, compared with no fatty acid addition (from 99.0+/-1.0% to 30.7+/-5.2% (p < 0.05) for DHA and 23.8+/-2.8% (p < 0.01) for EPA), while the saturated fatty acid, docosanoic acid (DA) and the methyl ester of DHA (DHA m.e.) did not exert a significant effect on asynchronous contractile activity. Asynchronous contractile activity was also reduced to 1.7+/-1.7% in the presence of the membrane fluidising agent, benzyl alcohol (p < 0.001 vs no fatty acid addition). Cell membrane fluidity was determined by steady state fluorescence anisotropy using the fluorescent probe, TMAP-DPH. Addition of DHA, EPA or benzyl alcohol significantly increased sarcolemmal membrane fluidity (decreased anisotropy, r(ss)) of atrial myocytes compared with no addition of fatty acid (control) (from r(ss) = 0.203+/-0.004 to 0.159+/-0.004 (p < 0.01) for DHA, 0.166+/-0.001 (p < 0.01) for EPA and 0.186+/-0.003 (p < 0.05) for benzyl alcohol, while DA and DHA m.e. were without effect. It is concluded that the n-3 PUFAs exert anti-asynchronous effects in rat atrial myocytes by a mechanism which may involve changes in membrane fluidity.

Young adult male Hooded Wistar rats were rendered diabetic by administration of streptozotocin and maintained for 5 weeks on a diet containing either 6% olive oil as the total source of fat (OO diet), or purified gamma-linolenic acid (GLA) at a concentration of 0.5% with the remaining 5.5% provided by olive oil (GLA diet). Rats were treated with the angiotensin converting inhibitor, cilazapril, administered in the drinking water at a dose of 20 mg kg-1 body weight day-1. For the OO diet groups, sciatic nerve conduction velocity (NCV) in diabetic rats was reduced by 32% (p < 0.01) in comparison with nondiabetic (vehicle-treated) rats and 27.5% (p < 0.05) in comparison with diabetic rats treated with cilazapril. Diabetic, cilazapril-treated rats showed no reduction in NCV. For the nondiabetic, diabetic, and diabetic plus cilazapril groups fed GLA, the NCV was not significantly different, indicating that dietary GLA also prevented the deficit in the NCV induced by the diabetic state. Analysis of the sciatic nerve endoneurial phospholipid fatty acids revealed a significant reduction in the proportion of GLA and an elevation in the proportion of linoleic acid in the diabetic groups compared with the nondiabetic groups and this was independent of the cilazapril treatment or the dietary lipid supplement. Sciatic nerve myo-inositol content was unaltered while mannose, fructose, glucose, and sorbitol levels were elevated in the diabetic groups and these changes were independent of the cilazapril treatment or the dietary lipid supplement. These results indicate that in the rat, cilazapril treatment or dietary GLA, at the doses tested, are effective in preventing the deficit in the NCV induced by diabetes.

Proton-dependent, ethylisopropylamiloride (EIPA)-sensitive Na+ uptake (Na+/H+ antiporter) studies were performed to examine if saliva, and ionophores which alter cellular electrolyte balance, could influence the activity of the cheek cell Na+/H+ antiporter. Using the standard conditions of 1 mmol/l Na+, and a 65:1 (inside:outside) proton gradient in the assay, the uniport ionophores valinomycin (K+) and gramicidin (Na+) increased EIPA-sensitive Na+ uptake by 177% (p < 0.01) and 227% (p < 0.01), respectively. The dual antiporter ionophore nigericin (K(+)-H+) increased EIPA-sensitive Na+ uptake by 654% (p < 0.01), with maximal Na+ uptake achieved by 1 min and at an ionophore concentration of 50 mumol/l, with an EC50 value 6.4 mumol/l. Pre-incubation of cheek cells with saliva or the low molecular weight (MW) components of saliva (saliva activating factors, SAF) for 2 h at 37 degrees C, also significantly stimulated EIPA-sensitive Na+ uptake. This stimulation could be mimicked by pre-incubation with 25 mmol/l KCl or K(+)-phosphate buffer. Pre-incubating cheek cells with SAF and the inclusion of 20 mumol/l nigericin in the assay, produced maximum EIPA-sensitive Na+ uptake. After pre-incubation with water, 25 mmol/l K(+)-phosphate or SAF, with nigericin in all assays, the initial rate of proton-gradient dependent, EIPA-sensitive Na+ uptake was saturable with respect to external Na+, with Km values of 0.9, 1.7, and 1.8 mmol/l, and Vmax values of 13.4, 25.8, and 31.1 nmol/mg protein/30 sec, respectively. With 20 mumol/l nigericin in the assay, Na+ uptake was inhibited by either increasing the [K+]o in the assay, with an ID50 of 3 mmol/l. These results indicate that nigericin can facilitate K+i exchange for H+o and the attending re-acidification of the cheek cell amplifies 22Na+ uptake via the Na+/H+ antiporter. The degree of stimulation of proton-dependent, EIPA-sensitive Na+ uptake is therefore dependent, in part, on the intracellular [K+]i.

Na+ transport activity was measured in cheek cells from untreated hypertensive subjects and age-matched normotensive controls identified from a blood pressure screening program. Cheek cells were isolated by a simple mouth wash procedure and Na+ transport activity was measured as the proton-dependent uptake of 22Na+ using a rapid filtration assay. The rate of Na+ uptake was about 45% lower in hypertensive subjects and this difference persisted in a follow up study 2 years later involving those subjects who remained untreated for their hypertension. The proton independent Na+ uptake was also reduced by about 46% in the hypertensive group. The increase in the rate of cheek cell Na+ transport with increasing transcellular proton gradient values was also significantly lower in hypertensive subjects. The reduced cheek cell Na+ transport observed in hypertensive subjects may indicate decreased activity of the Na+/H+ antiporter and/or changes in the ion permeability properties of the cheek cell plasma membrane in the hypertensive state. This novel assay provides a biochemically based method for discriminating between normotensive and hypertensive subjects and makes use of tissue which can be obtained in a relatively non-invasive manner.

Na+ transport activity was characterized in human cheek epithelial cells obtained from normotensive adult subjects. The cells were isolated using a mouth-wash procedure and assayed for Na+ uptake using a radioactive (22Na+) rapid filtration assay. Cheek cells displayed proton-dependent Na+ uptake activity that was dependent on the magnitude of the externally directed proton gradient measured using the fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to determine intracellular pH. Amiloride, ethylisopropylamiloride (EIPA), 5-(N,N-dimethyl)-amiloride, 5-(N-methyl-N-isobutyl)-amiloride (MIA), and 5-(N,N-hexamethylene)-amiloride (NNHA) all inhibited proton-dependent Na+ uptake, with MIA, EIPA, and NNHA being the most potent. The Michaelis constant (Km) for extracellular Na+ was 5.7 mM, while the maximum velocity for Na(+)-H+ antiporter activity was 4.3 nmol Na+.mg protein-1.30s-1. The Km for intracellular H+ was 0.17 microM, with a Hill coefficient of 0.7. Stimulation by ouabain and inhibition by bumetanide of cheek cell proton-dependent Na+ uptake indicated only relatively low activities of Na(+)-K(+)-ATPase and Na(+)-K(+)-2Cl- cotransport, respectively. These results are consistent with the presence of Na(+)-H+ antiporter activity in cheek cells. Cheek cells therefore provide a convenient, relatively noninvasive source of tissue for examining Na(+)-H+ antiporter activity in human subjects.

This study describes some biological properties of human cheek (buccal epithelial) cells, isolated by mouth wash. Yields ranged from 6.5 to 20.6 x 10(6) cells, with a mean (+/- SE) of 12.2 +/- 4.2 x 10(6) cells, which gave 0.55 +/- 0.01 x 10(6) cells/mg protein. Vital stain exclusion was similar in cells isolated in either water (89 +/- 2%) or 250 mM sucrose (87 +/- 3%). From our measurements of cell volume and electrolyte content, we estimated intracellular Na+ and K+ concentrations to be between 0.3-0.5 and 7.4-13.0 mM, respectively. In 22 adult subjects, basal, prickle, intermediate, and superficial cells represented 0.3 +/- 1.4, 51 +/- 2.4, 26 +/- 0.9, and 22.7 +/- 1.8%, respectively, of the total sample. Cheek cells exhibited a low endogenous rate of oxygen consumption, which was stimulated by glucose or succinate and inhibited by KCN or NaF. Cheek cells were osmotically stable in a wide range of media, including water. However, they exhibited shrinkage and collapse in hypertonic media, particularly polyethylene glycol.

Sodium transport including amiloride-sensitive Na+/H+ antiporter activity was measured in cheek epithelial cells of adolescents displaying either high or low BP tracking characteristics and in a subgroup of high BP tracking adolescents exhibiting a positive family history of hypertension. From the BP tracking behaviour of over 500 adolescents measured over a period of three years, 24 low BP tracking and 29 high BP tracking adolescents were recruited for the study. Cheek cells were collected from these subjects and proton-dependent, amiloride-sensitive Na+/H+ antiporter activity and the response of this antiporter to a proton gradient were measured. Cheek cell Na+/H+ antiporter activity was 50% lower (P = 0.0004) in the high BP tracking group (1.02 +/- 0.15 nmol Na+/mg protein/5 min (mean +/- SEM) compared with the activity in the low BP tracking group (2.05 +/- 0.24). A significantly lower Na+/H+ antiporter activity (69%; P < 0.01) was also apparent in the high BP tracking adolescents with family history of hypertension (n = 7) compared with the low BP tracking group. The graded response of cheek cell Na+/H+ antiporter activity to the proton gradient was 58% lower (P = 0.0039) for adolescents in the high BP tracking group compared with the low BP tracking group. Passive Na+ influx was also significantly lower in the cheek cells of the high BP tracking group. Our results therefore show that the activity of the Na+/H+ antiporter in cheek cells and the passive Na+ transport activity are lower in those adolescents considered at greatest risk of future development of essential hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously shown cheek cell Na+/H+ antiporter activity to be reduced in human hypertensives. We have now examined the relationship between abnormal antiporter activity and a variety of salivary factors. Total protein concentration and amylase activity were higher in hypertensives, but salivary flow rate and epidermal growth factor, transforming growth factor-alpha, calcium, and magnesium concentrations were not significantly different between hypertensives and normotensives. The lowered cheek cell Na+/H+ antiporter activity in those hypertensives with diastolic BP greater than 95 mmHg was accompanied by lowered salivary Na+/H+ ratios. In borderline hypertensives (diastolic BP between 90 and 95 mmHg), the Na+/H+ ratio was reduced to a similar extent to that seen in those hypertensives with a diastolic BP above 95 mmHg, however the cheek cell antiporter activity was not reduced, suggesting that these two differences are not related in a simple fashion in all hypertensives. It is concluded that it is unlikely that differences in salivary growth factors explain the lowered cheek cell Na+/H+ antiporter activity observed in human hypertension. Our findings indicate that salivary electrolyte composition may be related to cheek cell Na+/H+ antiporter activity and these parameters may be altered in hypertension.

Adult male marmoset monkeys were fed eicosapentaenoic acid (20:5n-3) as the ethyl ester in diets containing either 32% (reference diet, no added cholesterol) or 7% (atherogenic diet with 0.2% added cholesterol) linoleic acid (18:2n-6) for 30 wk. No changes were seen in the level of phosphatidylcholine (PC) or phosphatidylethanolamine (PE) but minor changes were observed in both the sphingomyelin (SPM) and phosphatidylinositol plus phosphatidylserine (PI+PS) fractions of erythrocyte lipids. The extent of total n-3 fatty acid incorporation into membrane lipids was higher in atherogenic diets (polyunsaturated/monounsaturated/saturated (P/M/S) ratio 0.2:0.6:1.0) than reference diets (P/M/S ratio 1:1:1) and this was true for both PE (33.4 +/- 1.0% vs 24.3 +/- 1.1%) and PC (9.3 +/- 0.5% vs 4.9 +/- 0.3%). Although suitable controls for cholesterol effects were not included in the study, earlier results obtained with marmosets lead us to believe such effects were probably small. Regardless of basic diet (atherogenic, reference), 20:5n-3 was preferentially incorporated into PE (10.8 +/- 0.2%, 6.0 +/- 0.02%) while smaller amounts were incorporated into PC (6.9 +/- 0.4%, 3.2 +/- 0.2%). The major n-3 polyunsaturated fatty acid found in PE in response to dietary 20:5n-3 was the elongation metabolite 22:5n-3 in both the atherogenic (17.7 +/- 0.7%) and reference (14.3 +/- 1.0%) dietary groups; 22:6n-3 levels were less affected by diet (4.7 +/- 0.3% and 3.9 +/- 0.2%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of dietary eicosapentaenoic acid (EPA, 20:5(n-3), as the ethyl ester) on plasma lipid levels and the incorporation of EPA into erythrocyte and plasma lipids were investigated in the marmoset monkey. Marmosets were fed high mixed-fat diets (14.5% total fat) supplemented with or without 0.8% EPA for 30 weeks. Markedly elevated plasma cholesterol (16.4 mmol/l) was induced by an atherogenic-type diet but with EPA supplementation, plasma cholesterol increased to only 6.6 mmol/l. Plasma triacylglycerol levels were not elevated with an atherogenic type diet. Substantial EPA incorporation was evident for plasma phospholipid, triacylglycerol and cholesterol ester fractions. The proportion of docosapentaenoic acid (22:5(n-3)) but not docosahexaenoic acid (22:6(n-3)) was also elevated in these plasma lipid fractions. Greatest incorporation of EPA occurred when it was administered with an atherogenic type diet having a P:M:S (polyunsaturated:monounsaturated:saturated) fatty acid ratio of about 0.2:0.6:1.0 in comparison to the control diet of 1.0:1.0:1.0. Incorporation of EPA and 22:5(n-3)) into erythrocyte phospholipids was also apparent and this was at the expense of linoleic acid (18:2(n-6)). These results in the marmoset highlight both the cholesterol-lowering properties of EPA and the extent of its incorporation into plasma lipids and erythrocyte membrane phospholipids with far greater incorporation occurring when the level of dietary linoleic acid was reduced.

Diets supplemented with high levels of saturated or unsaturated fatty acids supplied by addition of sheep kidney fat or sunflower seed oil, respectively, were fed to rats with or without dietary cholesterol. The effects of these diets on cardiac membrane lipid composition, catecholamine-stimulated adenylate cyclase and beta-adrenergic receptor activity associated with cardiac membranes, were determined. The fatty acid-supplemented diets, either with or without cholesterol, resulted in alterations in the proportion of the (n-6) to (n-3) series of unsaturated fatty acids, with the sunflower seed oil increasing and the sheep kidney fat decreasing this ratio, but did not by themselves significantly alter the ratio of saturated to unsaturated fatty acids. However, cholesterol supplementation resulted in a decrease in the proportion of saturated and polyunsaturated fatty acids and a dramatic increase in oleic acid in cardiac membrane phospholipids irrespective of the nature of the dietary fatty acid supplement. The cholesterol/phospholipid ratio of cardiac membrane lipids was also markedly increased with dietary cholesterol supplementation. Although relatively unaffected by the nature of the dietary fatty acid supplement, catecholamine-stimulated adenylate cyclase activity was significantly increased with dietary cholesterol supplementation and was positively correlated with the value of the membrane cholesterol/phospholipid ratio. Although the dissociation constant for the beta-adrenergic receptor, determined by [125I](-)-iodocyanopindolol binding, was unaffected by the nature of the dietary lipid supplement, the number of beta-adrenergic receptors was dramatically reduced by dietary cholesterol and negatively correlated with the value of the membrane cholesterol/phospholipid ratio. These results indicate that the activity of the membrane-associated beta-adrenergic/adenylate cyclase system of the heart can be influenced by dietary lipids particularly those altering the membrane cholesterol/phospholipid ratio and presumably membrane physico-chemical properties. In the face of these dietary-induced changes, a degree of homeostasis was apparent both with regard to membrane fatty acid composition in response to an altered membrane cholesterol/phospholipid ratio, and to down regulation of the beta-adrenergic receptor in response to enhanced catecholamine-stimulated adenylate cyclase activity.

Catecholamines and Ca2+ mediate an increase in reactive-thiol status of phosphofructokinase, associated with an increase in activation of the enzyme. A correlation was observed between the number of reactive thiols and activation (r = 0.878; P less than 0.001) for 14 different treatments, with approx. 1 group per 85,000-Mr subunit becoming available over the range from the lowest to the highest state of activation.

Ferricyanide was reduced to ferrocyanide by the perfused rat heart at a linear rate of 78 nmol/min per g of heart (non-recirculating mode). Ferricyanide was not taken up by the heart and ferrocyanide oxidation was minimal (3 nmol/min per g of heart). Perfusate samples from hearts perfused without ferricyanide did not reduce ferricyanide. A single high-affinity site (apparent Km = 22 microM) appeared to be responsible for the reduction. Perfusion of the heart with physiological medium containing 0.5 mM ferricyanide did not alter contractility, biochemical parameters or energy status of the heart. Perfusate flow rate and perfusate oxygen concentration exerted opposing effects on the rate of ferricyanide reduction. A net decreased reduction rate resulted from a decreased perfusion flow rate. Thus, the rate of supply of ferricyanide dominated over the stimulatory effect of oxygen restriction; the latter effect only becoming apparent when the oxygen concentration was lowered at a high perfusate flow rate. Whereas glucose (5 mM) increased the rate of ferricyanide reduction, pyruvate (2 mM), acetate (2 mM), lactate (2 mM) and 3-hydroxybutyrate (2 mM) each had no effect. Insulin (3 nM), glucagon (0.5 microM), dibutyryl cyclic AMP (0.1 mM) and the beta-adrenergic agonist ritodrine (10 microM) also had no effect, however, the alpha 1-adrenergic agonist, methoxamine (10 microM), produced a net increase in the rate of ferricyanide reduction. It is concluded that a trans-plasma membrane electron efflux occurs in perfused rat heart that is sensitive to oxygen supply, glucose, perfusion flow rate, and the alpha-adrenergic agonist methoxamine.

The activity of the beta-adrenergic receptor/adenylate cyclase system of the marmoset monkey heart was investigated following dietary cholesterol supplementation (0.5%). After 22 weeks, plasma cholesterol levels in the cholesterol group were more than twice that of the control group. In the cholesterol-fed group, the affinity for ICYP binding to cardiac membranes was elevated more than 2-fold, while the receptor number was decreased by 31%. Isoproterenol, norepinephrine and sodium fluoride stimulated adenylate cyclase activity was significantly higher in the cholesterol-fed group although the fold stimulation over basal levels was not affected. The most prominent change in the cardiac membrane lipids was an increase in the cholesterol to phospholipid ratio in marmoset monkeys fed cholesterol. These results indicate that in the marmoset, membrane cholesterol is an important factor in determining various properties of the cardiac beta-adrenergic receptor particularly receptor affinity which may impact on the response of the beta-adrenergic receptor/adenylate cyclase system of the heart to catecholamines. This result is in agreement with dietary fatty acid supplements designed to increase cardiac membrane cholesterol in this animal species (McMurchie, E.J. et al. (1988) Biochim. Biophys. Acta 937, 347-358). Elevated membrane cholesterol enhances beta-adrenergic receptor affinity and certain aspects of adenylate cyclase activity. This is a likely mechanism whereby atherogenic diets could promote cardiac arrhythmia in non-human primates and indeed in man.

Perfusion of the isolated rat heart with Ca2+ concentrations exceeding 3 mM activated phosphofructokinase and phosphorylase, and decreased the concentration of cyclic AMP. Half-maximal activation of phosphofructokinase occurred at 5 mM-CaCl2; significant activation of phosphorylase did not occur until the concentration of CaCl2 exceeded 12 mM. The time course for the activation of phosphofructokinase at 12 mM-CaCl2 indicated that maximal activation occurred within 2 min; when the perfusion-medium Ca2+ concentration was re-adjusted to 3 mM, the phosphofructokinase activity returned to pre-activation values within 30 s. The addition of Ca2+ to extracts of heart did not activate phosphofructokinase. The activation of phosphofructokinase by sub-maximal doses of adrenaline and Ca2+ were not additive. The activation of phosphofructokinase by 1 microM-adrenaline + 10 microM-propranolol and by 1 microM-isoprenaline was inhibited by high concentrations of K+ (22-56 mM). The activation of phosphofructokinase by 1 microM-adrenaline + 10 microM-propranolol, 12 mM-CaCl2 and by 1 microM-isoprenaline was blocked by the slow Ca2+-channel blocker nifedipine. These findings suggest that both the beta- and alpha-adrenergic mechanisms for the activation of rat heart phosphofructokinase involve an increase in the myoplasmic Ca2+ concentration. This increase may result from an inhibition of Ca2+ efflux or a stimulation of Ca2+ influx.

Epinephrine treatment of the perfused rat heart led to an increase in glucose uptake, detritiation of [5-3H] glucose, glycogenolysis, and the formation of lactate. The change in the rate of formation of 3H2O from [5-3H]glucose was slower to develop (commencing at approximately 30 s) than changes in cyclic AMP concentration, hexose-6-P concentration, and the phosphorylase a/(a + b) ratio which were maximal at 24 s. Epinephrine plus propranolol (alpha-adrenergic combination) treatment of the perfused heart also led to increases in glucose uptake, detritiation of [5-3H]glucose, and the formation of lactate, but these occurred without significant changes in cyclic AMP concentration, hexose-6-P concentration, or the phosphorylase a/(a + b) ratio. Half-maximal stimulation of glucose uptake occurred at 0.2 microM epinephrine, 1.5 microM methoxamine, and 1 microM isoproterenol. The increase in glucose uptake mediated by 1 microM epinephrine was blocked by 10 microM prazosin but unaffected by 10 microM propranolol. The increase in glucose uptake mediated by 10 microM epinephrine plus 10 microM propranolol was partly blocked by yohimbine and completely blocked by prazosin. A role for Ca2+ in the adrenergic regulation of glucose uptake was indicated by the sensitivity of the epinephrine dose curve to Ca2+ and the dependence of epinephrine on Ca2+. In addition the increases in glucose uptake mediated by 1 microM epinephrine, 1 microM epinephrine plus 10 microM propranolol, 1 microM isoproterenol, and by 10 mM CaCl2 were each blocked by the Ca2+ channel blocker nifedipine (1 microM). It is concluded that Ca2+-dependent alpha- and beta-adrenergic receptor mechanisms are present in rat heart for controlling glucose uptake. At submicromolar levels of epinephrine the predominant receptors utilized appear to be alpha 1.

Phosphofructokinase was determined at low substrate concentration using a new isotopic assay in extracts of perfused rat heart. Epinephrine treatment of the perfused heart resulted in an activation of the enzyme. Half-maximal activation of phosphofructokinase occurred at 5 X 10(-7) M epinephrine, which was approximately that required to produce half-maximal activation of phosphorylase. Time course studies indicated that epinephrine-mediated changes in beating rate, cyclic AMP concentration, and phosphorylase a activity were maximal at 1 to 2 min and preceded maximal activation of phosphofructokinase by approximately 3 min. the activated form of the enzyme as expressed in heart extracts was sensitized to the activators, cyclic AMP, AMP, glucose 1,6-bisphosphate, and fructose 1,6-bisphosphate. Passage of control extract that was untreated, activated by AMP, or inhibited by citrate through Sephadex G-25 columns gave eluate activities approaching control extract values. The epinephrine-activated form of the enzyme remained activated following similar treatment. The data suggest that epinephrine mediates a modification of phosphofructokinase that is independent of changes in intracellular effector concentration.

Eicosapentaenoic acid (EPA; 20:5 n-3; ethyl ester) in combination with atherogenic or non-atherogenic high fat diets was fed to young adult male marmoset monkeys for a period of 30 weeks. EPA markedly reduced the raised plasma cholesterol level evident when feeding an atherogenic diet but did not influence the cardiac membrane cholesterol-to-phospholipid ratio. EPA and its elongation product 22:5 n-3 was incorporated into cardiac membrane phospholipids at the expense of linoleic and arachidonic acids. Dietary EPA increased cardiac beta-AR affinity and reversed the decreased beta-AR affinity evident when feeding an atherogenic diet.

1. A comparison was made between adrenergic receptor binding properties and catecholamine-stimulated adenylyl cyclase activity in cardiac membrane fractions from the rat and the marmoset monkey. 2. [125I]HEAT and [125I]ICYP were used to determine respectively, the alpha- and beta-adrenergic receptor binding in cardiac membrane fractions. 3. Greatest adrenergic receptor density and degree of specific binding was evident using membranes sedimenting between 6000 and 46,000 g. 4. In rat heart, the ratio of beta- to alpha-adrenergic receptors was 57:43, while for the marmoset this ratio was 92:8. 5. Basal, isoproterenol, sodium fluoride and forskolin-stimulated adenylyl cyclase activities in the rat and marmoset monkey were investigated in several different cardiac membrane fractions. 6. The highest-fold stimulation of adenylyl cyclase activity was present in membranes sedimenting between 0 and 500 g. 7. Adenylyl cyclase activities were higher in the marmoset heart membrane preparations, however the rat heart adenylyl cyclase exhibited greater sensitivity to isoproterenol; ED50 3.8 X 10(-7) M compared with 7.5 X 10(-7) M for the marmoset. 8. Differences between rat and marmoset catecholamine-sensitive adenylyl cyclase activity were apparent when a variety of adrenergic agonists and antagonists were tested. 9. In the marmoset but not the rat, adrenergic antagonists alone stimulated basal adenylyl cyclase activity. 10. Differences in the activation of cardiac adenylyl cyclase by GTP and GMP-PNP were also evident between the rat and the marmoset monkey, particularly with regard to basal and isoproterenol-stimulated activity.

1. Reduction of ferricyanide by the isolated perfused rat liver and by isolated rat hepatocytes was studied. 2. Ferricyanide was reduced to ferrocyanide by the perfused liver at a linear rate of 0.22mumol/min per g of liver. Ferricyanide was not taken up by the liver and the perfusate concentration of ferricyanide+ferrocyanide remained constant throughout the perfusion. Perfusate samples from livers perfused without ferricyanide did not reduce ferricyanide. 3. Isolated hepatocytes reduced ferricyanide in a biphasic manner. The initial rate of 2.3mumol/min per g of cells proceeded for approx. 3min and derived from low-affinity sites (apparent K(m)>1.3mm). The secondary rate of 0.29mumol/min per g of cells was maintained for the remainder of the incubation and derived from higher affinity sites (apparent K(m)0.13mm). Disruption of the cells resulted in an increase in the low-affinity rate and a decrease in the high-affinity rate. 4. Ferrocyanide was oxidized by isolated hepatocytes but not by perfused liver. The apparent K(m) for ferrocyanide oxidation by hepatocytes was 1.3mm. 5. Oxidized cytochrome c was reduced by isolated hepatocytes in the presence of 1mm-KCN but at a rate less than that of the reduction of ferricyanide. 6. Properties of the ferricyanide-reducing activities of intact hepatocytes and the perfused liver were examined. The low-affinity rate, present only in cell and broken cell preparations, was inhibited by 1mum-rotenone and 0.5mm-ferrocyanide, and stimulated by 0.1mm-KCN. The mitochondrial substrate, succinate, also stimulated this rate. The perfused liver showed only a high-affinity activity for ferricyanide reduction. This activity was also present in liver cells and was unaffected by rotenone, antimycin A, KCN, NaN(3), or p-hydroxymercuribenzoate but was inhibited by 2.6mm-CaCl(2), 2-heptyl-4-hydroxyquinoline-N-oxide and ferrocyanide. Overall, these results are consistent with the occurrence of a trans-plasma membrane redox system of liver that reduces extracellular ferricyanide to ferrocyanide. The reduction process shows properties which are similar to that of the NADH:ferricyanide oxidoreductase found in isolated liver plasma membranes but different from that of mitochondria.

The ascending and descending relationships of visual cortex in Trichosurus were determined using HRP and autoradiographic methods. The visual thalamus, LGNd and LP, was found to project to three cytoarchitecturally distinct areas of cortex with each of these regions displaying differing patterns of connectivity with other centres. The striate and peristriate cortices received homotypically organized projections from LGNd as well as projections from both divisions of LP. The posterior parietal cortex received projections from both LGNd and LP as well as from the latero-intermediate, posterior, ventroanterior and intralaminar thalamic nuclei. The cortical projection fields of LGNd and LP were co-extensive. Descending projections to thalamic centres were reciprocal with the exception of a descending only projection to the thalamic reticular nucleus. The striatogeniculate projection was homotypically organized as was the striatocollicular projection which extended only to the superficial layers of that nucleus. The posterior parietal projection to the superior colliculus involved the deeper layers of that nucleus. The entire visual cortex projected to the pretectum and to the pontine nuclei. The organization of visual cortex in Trichosurus differs from that reported in the American marsupial, Didelphis, in that the LGNd and LP complexes possess a very broad and co-extensive cortical projection, a finding substantially different to what has been reported in placental mammals as well. The organization of thalamic relationships with the posterior parietal cortex would also appear to be unusual with this region receiving convergent projections from at least seven separate thalamic nuclei.

Recent findings indicate that glucose uptake by contracting hindlimb #Acta Physiol. Scand. (1982) 116, 215-222 #and heart #Biochem. Biophys. Res. Commun. (1982) 108, 124-131 # of the rat is stimulated by epinephrine acting through alpha-adrenergic mechanisms. Since in exercise hepatic glucose output may be increased markedly by activation of alpha-adrenergic receptors and matched by the increase in muscle glucose uptake (maintaining blood glucose levels relatively constant), it is now proposed that a general coordination of glucose metabolism may operate via alpha-adrenergic receptor mechanisms. The basis for this proposal is discussed.