Dapagliflozin reduces the amplitude of shortening and Ca(2+) transient in ventricular myocytes from streptozotocin-induced diabetic rats.

In the management of type 2 diabetes mellitus, Dapagliflozin (DAPA) is a newly introduced selective sodium-glucose co-transporter 2 inhibitor which promotes renal glucose excretion. Little is known about the effects of DAPA on the electromechanical function of the heart. This study investigated the effects of DAPA on ventricular myocyte shortening and intracellular Ca(2+) transport in streptozotocin (STZ)-induced diabetic rats. Shortening, Ca(2+) transients, myofilament sensitivity to Ca(2+) and sarcoplasmic reticulum Ca(2+), and intracellular Ca(2+) current were measured in isolated rats ventricular myocytes by video edge detection, fluorescence photometry, and whole-cell patch-clamp techniques. Diabetes was characterized in STZ-treated rats by a fourfold increase in blood glucose (440 ± 25 mg/dl, n = 21) compared to Controls (98 ± 2 mg/dl, n = 19). DAPA reduced the amplitude of shortening in Control (76.68 ± 2.28 %, n = 37) and STZ (76.58 ± 1.89 %, n = 42) ventricular myocytes, and reduced the amplitude of the Ca(2+) transients in Control and STZ ventricular myocytes with greater effects in STZ (71.45 ± 5.35 %, n = 16) myocytes compared to Controls (92.01 ± 2.72 %, n = 17). Myofilament sensitivity to Ca(2+) and sarcoplasmic reticulum Ca(2+) were not significantly altered by DAPA in either STZ or Control myocytes. L-type Ca(2+) current was reduced in STZ myocytes compared to Controls and was further reduced by DAPA. In conclusion, alterations in the mechanism(s) of Ca(2+) transport may partly underlie the negative inotropic effects of DAPA in ventricular myocytes from STZ-treated and Control rats.

Effects of a sucrose-enriched diet on the pattern of gene expression, contraction and Ca(2+) transport in Goto-Kakizaki type 2 diabetic rat heart.

There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus (T2DM), and cardiovascular disease is the major cause of morbidity and mortality in diabetic patients. A variety of diastolic and systolic dysfunctions have been demonstrated in type 2 diabetic heart. The consumption of sugar-sweetened beverages has been linked to rising rates of obesity, which in turn is a risk factor for development of T2DM. In this study, the effects of a sucrose-enriched diet on the pattern of gene expression, contraction and Ca(2+) transport in the Goto-Kakizaki T2DM rat heart were investigated. Genes encoding cardiac muscle proteins (Myh7, Mybpc3, Myl1, Myl3 and Mylpf), intercellular proteins (Gja4), cell membrane transport (Atp1b1), calcium channels (Cacna1c, Cacna1g and Cacnb1) and potassium channels (Kcnj11) were upregulated and genes encoding potassium channels (Kcnb1) were downregulated in GK compared with control rats. Genes encoding cardiac muscle proteins (Myh6, Mybpc3 and Tnn2), intercellular proteins (Gja1 and Gja4), intracellular Ca(2+) transport (Atp2a1 and Ryr2), cell membrane transport (Atp1a2 and Atp1b1) and potassium channel proteins (Kcnj2 and Kcnj8) were upregulated and genes encoding cardiac muscle proteins (Myh7) were downregulated in control rats fed sucrose compared with control rats. Genes encoding cardiac muscle proteins (Myh7) and potassium channel proteins (Kcnj11) were downregulated in control and GK rats fed sucrose compared with control and GK rats, respectively. The amplitude of shortening was reduced in myocytes from the control-sucrose group compared with control rats and in the GK-sucrose group compared with GK rats. The amplitude of the Ca(2+) transient was increased in myocytes from control-sucrose compared with control rats and decreased in GK-sucrose compared with GK rats. Subtle alterations in the pattern of expression of genes encoding a variety of cardiac muscle proteins are associated with changes in shortening and intracellular Ca(2+) transport in ventricular myocytes from GK T2DM and control rats fed a sucrose-enriched diet.

Effects of exercise training on excitation-contraction coupling and related mRNA expression in hearts of Goto-Kakizaki type 2 diabetic rats.

Although, several novel forms of intervention aiming at newly identified therapeutic targets are currently being developed for diabetes mellitus (DM), it is well established that physical exercise continues to be one of the most valuable forms of non-pharmacological therapy. The aim of the study was to investigate the effects of exercise training on excitation-contraction coupling and related gene expression in the Goto-Kakizaki (GK) type 2 diabetic rat heart and whether exercise is able to reverse diabetes-induced changes in excitation-contraction coupling and gene expression. Experiments were performed in GK and control rats aged 10-11 months following 2-3 months of treadmill exercise training. Shortening, [Ca(2+)]i and L-type Ca(2+) current were measured in ventricular myocytes with video edge detection, fluorescence photometry and whole cell patch clamp techniques, respectively. Expression of mRNA was assessed in ventricular muscle with real-time RT-PCR. Amplitude of shortening, Ca(2+) transients and L-type Ca(2+) current were not significantly altered in ventricular myocytes from GK sedentary compared to control sedentary rats or by exercise training. Expression of mRNA encoding Tpm2, Gja4, Atp1b1, Cacna1g, Cacnb2, Hcn2, Kcna3 and Kcne1 were up-regulated and Gja1, Kcnj2 and Kcnk3 were down-regulated in hearts of sedentary GK rats compared to sedentary controls. Gja1, Cav3 and Kcnk3 were up-regulated and Hcn2 was down-regulated in hearts of exercise trained GK compared to sedentary GK controls. Ventricular myocyte shortening and Ca(2+) transport were generally well preserved despite alterations in the profile of expression of mRNA encoding a variety of cardiac muscle proteins in the adult exercise trained GK diabetic rat heart.

Shortening and intracellular Ca2+ in ventricular myocytes and expression of genes encoding cardiac muscle proteins in early onset type 2 diabetic Goto-Kakizaki rats.

There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus. Cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. Contractile dysfunction, associated with disturbances in excitation-contraction coupling, has been widely demonstrated in the diabetic heart. The aim of this study was to investigate the pattern of cardiac muscle genes that are involved in the process of excitation-contraction coupling in the hearts of early onset (8-10 weeks of age) type 2 diabetic Goto-Kakizaki (GK) rats. Gene expression was assessed in ventricular muscle with real-time RT-PCR; shortening and intracellular Ca(2+) were measured in ventricular myocytes with video edge detection and fluorescence photometry, respectively. The general characteristics of the GK rats included elevated fasting and non-fasting blood glucose and blood glucose at 120 min following a glucose challenge. Expression of genes encoding cardiac muscle proteins (Myh6/7, Mybpc3, Myl1/3, Actc1, Tnni3, Tnn2, Tpm1/2/4 and Dbi) and intercellular proteins (Gja1/4/5/7, Dsp and Cav1/3) were unaltered in GK ventricle compared with control ventricle. The expression of genes encoding some membrane pumps and exchange proteins was unaltered (Atp1a1/2, Atp1b1 and Slc8a1), whilst others were either upregulated (Atp1a3, relative expression 2.61 ± 0.69 versus 0.84 ± 0.23) or downregulated (Slc9a1, 0.62 ± 0.07 versus 1.08 ± 0.08) in GK ventricle compared with control ventricle. The expression of genes encoding some calcium (Cacna1c/1g, Cacna2d1/2d2 and Cacnb1/b2), sodium (Scn5a) and potassium channels (Kcna3/5, Kcnj3/5/8/11/12, Kchip2, Kcnab1, Kcnb1, Kcnd1/2/3, Kcne1/4, Kcnq1, Kcng2, Kcnh2, Kcnk3 and Kcnn2) were unaltered, whilst others were either upregulated (Cacna1h, 0.95 ± 0.16 versus 0.47 ± 0.09; Scn1b, 1.84 ± 0.16 versus 1.11 ± 0.11; and Hcn2, 1.55 ± 0.15 versus 1.03 ± 0.08) or downregulated (Hcn4, 0.16 ± 0.03 versus 0.37 ± 0.08; Kcna2, 0.35 ± 0.03 versus 0.80 ± 0.11; Kcna4, 0.79 ± 0.25 versus 1.90 ± 0.26; and Kcnj2, 0.52 ± 0.07 versus 0.78 ± 0.08) in GK ventricle compared with control ventricle. The amplitude of ventricular myocyte shortening and the intracellular Ca(2+) transient were unaltered; however, the time-to-peak shortening was prolonged and time-to-half decay of the Ca(2+) transient was shortened in GK myocytes compared with control myocytes. The results of this study demonstrate changes in expression of genes encoding various excitation-contraction coupling proteins that are associated with disturbances in myocyte shortening and intracellular Ca(2+) transport.

Contractility of ventricular myocytes is well preserved despite altered mechanisms of Ca2+ transport and a changing pattern of mRNA in aged type 2 Zucker diabetic fatty rat heart.

There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus and cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. The objective of the study was to investigate ventricular myocyte shortening, intracellular Ca(2+) signalling and expression of genes encoding cardiac muscle proteins in the aged Zucker diabetic fatty (ZDF) rat. There was a fourfold elevation in non-fasting blood glucose in ZDF rats (478.43 ± 29.22 mg/dl) compared to controls (108.22 ± 2.52 mg/dl). Amplitude of shortening, time to peak (TPK) and time to half (THALF) relaxation of shortening were unaltered in ZDF myocytes compared to age-matched controls. Amplitude and THALF decay of the Ca(2+) transient were unaltered; however, TPK Ca(2+) transient was prolonged in ZDF myocytes (70.0 ± 3.2 ms) compared to controls (58.4 ± 2.3 ms). Amplitude of the L-type Ca(2+) current was reduced across a wide range of test potentials (-30 to +40 mV) in ZDF myocytes compared to controls. Sarcoplasmic reticulum Ca(2+) content was unaltered in ZDF myocytes compared to controls. Expression of genes encoding cardiac muscle proteins, membrane Ca(2+) channels, and cell membrane ion transport and intracellular Ca(2+) transport proteins were variously altered. Myh6, Tnnt2, Cacna2d3, Slc9a1, and Atp2a2 were downregulated while Myl2, Cacna1g, Cacna1h, and Atp2a1 were upregulated in ZDF ventricle compared to controls. The results of this study have demonstrated that preserved ventricular myocyte shortening is associated with altered mechanisms of Ca(2+) transport and a changing pattern of genes encoding a variety of Ca(2+) signalling and cardiac muscle proteins in aged ZDF rat.

Changing pattern of gene expression is associated with ventricular myocyte dysfunction and altered mechanisms of Ca2+ signalling in young type 2 Zucker diabetic fatty rat heart.

The association between type 2 diabetes and obesity is very strong, and cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. The aim of this study was to investigate early changes in the pattern of genes encoding cardiac muscle regulatory proteins and associated changes in ventricular myocyte contraction and Ca(2+) transport in young (9- to 13-week-old) type 2 Zucker diabetic fatty (ZDF) rats. The amplitude of myocyte shortening was unaltered; however, time-to-peak shortening and time to half-relaxation of shortening were prolonged in ZDF myocytes (163 ± 5 and 127 ± 7 ms, respectively) compared with age-matched control rats (136 ± 5 and 103 ± 4 ms, respectively). The amplitude of the Ca(2+) transient was unaltered; however, time-to-peak Ca(2+) transient was prolonged in ZDF myocytes (66.9 ± 2.6 ms) compared with control myocytes (57.6 ± 2.3 ms). The L-type Ca(2+) current was reduced, and inactivation was prolonged over a range of test potentials in ZDF myocytes. At 0 mV, the density of L-type Ca(2+) current was 1.19 ± 0.28 pA pF(-1) in ZDF myocytes compared with 2.42 ± 0.40 pA pF(-1) in control myocytes. Sarcoplasmic reticulum Ca(2+) content, release and uptake and myofilament sensitivity to Ca(2+) were unaltered in ZDF myocytes compared with control myocytes. Expression of genes encoding various L-type Ca(2+) channel proteins (Cacna1c, Cacna1g, Cacna1h and Cacna2d1) and cardiac muscle proteins (Myh7) were upregulated, and genes encoding intracellular Ca(2+) transport regulatory proteins (Atp2a2 and Calm1) and some cardiac muscle proteins (Myh6, Myl2, Actc1, Tnni3, Tnn2, and Tnnc1) were downregulated in ZDF heart compared with control heart. A change in the expression of genes encoding myosin heavy chain and L-type Ca(2+) channel proteins might partly underlie alterations in the time course of contraction and Ca(2+) transients in ventricular myocytes from ZDF rats.

Altered gene expression may underlie prolonged duration of the QT interval and ventricular action potential in streptozotocin-induced diabetic rat heart.

Ventricular electrical conduction has been investigated in the streptozotocin (STZ)-induced diabetic rat. Diabetes was induced with a single injection of STZ (60 mg/kg bodyweight, ip). The ECG was measured continuously, in vivo, using a biotelemetry system. Left ventricular action potentials were recorded with an extracellular suction electrode. Expression of mRNA transcripts for selected ion transport proteins was measured in left ventricle with real-time RT-PCR. At 10 weeks after STZ treatment, in vivo heart rate (HR) was reduced (267 +/- 3 vs. 329 +/- 5 BPM), QRS complex duration and QT interval were prolonged in diabetic rats compared to controls. In vitro spontaneous HR was reduced and paced heart action potential repolarization was prolonged in diabetic rats compared to controls. The mRNA expression for Kcnd2 (I (to) channel) and Kcne2 (I (kr) channel) was significantly reduced in diabetic rats compared to controls. Altered gene expression and, in particular, genes that encode K(+) channel proteins may underlie delayed propagation of electrical activity in the ventricular myocardium of STZ-induced diabetic rat.

Effects of streptozotocin-induced diabetes on connexin43 mRNA and protein expression in ventricular muscle.

Abnormal QT prolongation with the associated arrhythmias is a significant predictor of mortality in diabetic patients. Gap junctional intercellular communication allows electrical coupling between heart muscle cells. The effects of streptozotocin (STZ)-induced diabetes mellitus on the expression and distribution of connexin 43 (Cx43) in ventricular muscle have been investigated. Cx43 mRNA expression was measured in ventricular muscle by quantitative PCR. The distribution of total Cx43, phosphorylated Cx43 (at serine 368) and non-phosphorylated Cx43 was measured in ventricular myocytes and ventricular muscle by immunocytochemistry and confocal microscopy. There was no significant difference in Cx43 mRNA between diabetic rat ventricle and controls. Total and phosphorylated Cx43 were significantly increased in ventricular myocytes and ventricular muscle and dephosphorylated Cx43 was not significantly altered in ventricular muscle from diabetic rat hearts compared to controls. Disturbances in gap junctional intercellular communication, which in turn may be attributed to alterations in balance between total, phosphorylated and dephosporylated Cx43, might partly underlie prolongation of QRS and QT intervals in diabetic heart.

Effects of pioglitazone on ventricular myocyte shortening and Ca(2+) transport in the Goto-Kakizaki type 2 diabetic rat.

Pioglitazone (PIO) is a thiazolidindione antidiabetic agent which improves insulin sensitivity and reduces blood glucose in experimental animals and treated patients. At the cellular level the actions of PIO in diabetic heart are poorly understood. A previous study has demonstrated shortened action potential duration and inhibition of a variety of transmembrane currents including L-type Ca(2+) current in normal canine ventricular myocytes. The effects of PIO on shortening and calcium transport in ventricular myocytes from the Goto-Kakizaki (GK) type 2 diabetic rat have been investigated. 10 min exposure to PIO (0.1-10 microM) reduced the amplitude of shortening to similar extents in ventricular myocytes from GK and control rats. 1 microM PIO reduced the amplitude of the Ca(2+) transients to similar extents in ventricular myocytes from GK and control rats. Caffeine-induced Ca(2+) release from the sarcoplasmic reticulum and recovery of Ca(2+) transients following application of caffeine and myofilament sensitivity to Ca(2+) were not significantly altered in ventricular myocytes from GK and control rats. Amplitude of L-type Ca(2+) current was not significantly decreased in myocytes from GK compared to control rats and by PIO treatment. The negative inotropic effects of PIO may be attributed to a reduction in the amplitude of the Ca(2+) transient however, the mechanisms remain to be resolved.

Therapeutic potential of benfotiamine and its molecular targets.

OBJECTIVE: The water-soluble vitamin, thiamine forms an important part of the diet because of its role in the energy metabolism. The protective effects of thiamine against diabetic vascular complications have been well documented. However, slower absorption and reduced bioavailability is a major limiting factor for its clinical use. To overcome this issue, lipid-soluble derivatives of thiamine (allithiamines) was developed. Among the many synthetic lipophilic derivatives of thiamine, benfotiamine (BFT) is regarded as the first choice based on its safety and clinical efficacy data. BFT facilitates the action of thiamine diphosphate, a cofactor for the enzyme transketolase. The activation of transketolase enzyme accelerates the precursors of advanced glycation end products (AGEs) towards the pentose phosphate pathway thereby reducing the production of AGEs. The reduction in AGEs subsequently decreases metabolic stress which benefits vascular complications seen in diabetes. The effects of BFT on the AGE-dependent pathway is well established. However, several studies have shown that BFT also modulates pathways other than AGE such as arachidonic acid (AA), nuclear transcription Factor κB (NF-κß), protein kinase B, mitogen-activated protein kinases (MAPK) and vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathways. In the present review, we have comprehensively reviewed all the molecular targets modulated by BFT to provide mechanistic perspective to highlight its pleiotropic effects.

The Pattern of mRNA Expression Is Changed in Sinoatrial Node from Goto-Kakizaki Type 2 Diabetic Rat Heart.

BACKGROUND: In vivo experiments in Goto-Kakizaki (GK) type 2 diabetic rats have demonstrated reductions in heart rate from a young age. The expression of genes encoding more than 70 proteins that are associated with the generation and conduction of electrical activity in the GK sinoatrial node (SAN) have been evaluated to further clarify the molecular basis of the low heart rate. MATERIALS AND METHODS: Heart rate and expression of genes were evaluated with an extracellular electrode and real-time RT-PCR, respectively. Rats aged 12-13 months were employed in these experiments. RESULTS: Isolated spontaneous heart rate was reduced in GK heart (161 ± 12 bpm) compared to controls (229 ± 11 bpm). There were many differences in expression of mRNA, and some of these differences were of particular interest. Compared to control SAN, expression of some genes were downregulated in GK-SAN: gap junction, Gja1 (Cx43), Gja5 (Cx40), Gjc1 (Cx45), and Gjd3 (Cx31.9); cell membrane transport, Trpc1 (TRPC1) and Trpc6 (TRPC6); hyperpolarization-activated cyclic nucleotide-gated channels, Hcn1 (HCN1) and Hcn4 (HCN4); calcium channels, Cacna1d (Cav1.3), Cacna1g (Cav3.1), Cacna1h (Cav3.2), Cacna2d1 (Cavα2δ1), Cacna2d3 (Cavα2δ3), and Cacng4 (Cav γ 4); and potassium channels, Kcna2 (Kv1.2), Kcna4 (Kv1.4), Kcna5 (Kv1.5), Kcnb1 (Kv2.1), Kcnd3 (Kv4.3), Kcnj2 (Kir2.1), Kcnk1 (TWIK1), Kcnk5 (K2P5.1), Kcnk6 (TWIK2), and Kcnn2 (SK2) whilst others were upregulated in GK-SAN: Ryr2 (RYR2) and Nppb (BNP). CONCLUSIONS: This study provides new insight into the changing expression of genes in the sinoatrial node of diabetic heart.

Calcium Signaling in the Ventricular Myocardium of the Goto-Kakizaki Type 2 Diabetic Rat.

The association between diabetes mellitus (DM) and high mortality linked to cardiovascular disease (CVD) is a major concern worldwide. Clinical and preclinical studies have demonstrated a variety of diastolic and systolic dysfunctions in patients with type 2 diabetes mellitus (T2DM) with the severity of abnormalities depending on the patients' age and duration of diabetes. The cellular basis of hemodynamic dysfunction in a type 2 diabetic heart is still not well understood. The aim of this review is to evaluate our current understanding of contractile dysfunction and disturbances of Ca2+ transport in the Goto-Kakizaki (GK) diabetic rat heart. The GK rat is a widely used nonobese, nonhypertensive genetic model of T2DM which is characterized by insulin resistance, elevated blood glucose, alterations in blood lipid profile, and cardiac dysfunction.

Reduction in the amplitude of shortening and Ca(2+) transient by phlorizin and quercetin-3-O-glucoside in ventricular myocytes from streptozotocin-induced diabetic rats.

Diabetes mellitus is the leading cause of cardiovascular morbidity and mortality. Phlorizin (PHLOR) and quercetin-3-O-glucoside (QUER-3-G) are two natural compounds reported to have antidiabetic properties by inhibiting sodium/glucose transporters. Their effects on ventricular myocyte shortening and intracellular Ca(2+) in streptozotocin (STZ)-induced diabetic rats were investigated. Video edge detection and fluorescence photometry were used to measure ventricular myocyte shortening and intracellular Ca(2+), respectively. Blood glucose in STZ rats was 4-fold higher (469.64+/-22.23 mg/dl, n=14) than in Controls (104.06+/-3.36 mg/dl, n=16). The amplitude of shortening was reduced by PHLOR in STZ (84.76+/-2.91 %, n=20) and Control (83.72+/-2.65 %, n=23) myocytes, and by QUER-3-G in STZ (79.12+/-2.28 %, n=20) and Control (76.69+/-1.92 %, n=30) myocytes. The amplitude of intracellular Ca(2+) was also reduced by PHLOR in STZ (82.37+/-3.16 %, n=16) and Control (73.94+/-5.22 %, n=21) myocytes, and by QUER-3-G in STZ (73.62+/-5.83 %, n=18) and Control (78.32+/-3.54 %, n=41) myocytes. Myofilament sensitivity to Ca(2+) was not significantly altered by PHLOR; however, it was reduced by QUER-3-G modestly in STZ myocytes and significantly in Controls. PHLOR and QUER-3-G did not significantly alter sarcoplasmic reticulum Ca(2+) in STZ or Control myocytes. Altered mechanisms of Ca(2+) transport partly underlie PHLOR and QUER-3-G negative inotropic effects in ventricular myocytes from STZ and Control rats.

Distribution of atrial natriuretic peptide and its effects on contraction and intracellular calcium in ventricular myocytes from streptozotocin-induced diabetic rat.

The distribution of atrial natriuretic peptide (ANP) in blood plasma and cardiac muscle and its effects on ventricular myocyte contraction and intracellular free calcium concentration [Ca2+]i in the streptozotocin (STZ)-induced diabetic rat have been investigated. Blood plasma concentration and heart atrial and ventricular contents of ANP were significantly increased in STZ-treated rats compared to age-matched controls. STZ treatment increased the number of ventricular myocytes immunolabeled with antibodies against ANP. In control myocytes the percentage of cells that labeled positively and negatively were 17% versus 83%, respectively. However, in myocytes from STZ-treated rat the percentages were 52% versus 53%. Time to peak (TPK) shortening was significantly and characteristically prolonged in myocytes from STZ-treated rats (360+/-5 ms) compared to controls (305+/-5 ms). Amplitude of the Ca2+ transient was significantly increased in myocytes from STZ-treated rats compared to controls (0.39+/-0.02 versus 0.29+/-0.02 fura-2 RU in controls) and treatment with ANP reduced the amplitude of the Ca2+ transient to control levels. ANP may have a protective role in STZ-induced diabetic rat heart.

Inhibitory actions of bisabolol on α7-nicotinic acetylcholine receptors.

Bisabolol is a plant-derived monocyclic sesquiterpene alcohol with antinociceptive and antiinflammatory actions. However, molecular targets mediating these effects of bisabolol are poorly understood. In this study, using a two-electrode voltage-clamp and patch-clamp techniques and live cellular calcium imaging, we have investigated the effect of bisabolol on the function of human α7 subunit of nicotinic acetylcholine receptor (nAChR) in Xenopus oocytes, interneurons of rat hippocampal slices. We have found that bisabolol reversibly and concentration dependently (IC50 = 3.1 µM) inhibits acetylcholine (ACh)-induced α7 receptor-mediated currents. The effect of bisabolol was not dependent on the membrane potential. Bisabolol inhibition was not changed by intracellular injection of the Ca(2+) chelator BAPTA and perfusion with Ca(2+)-free solution containing Ba(2+), suggesting that endogenous Ca(2+)-dependent Cl(-) channels are not involved in bisabolol actions. Increasing the concentrations of ACh did not reverse bisabolol inhibition. Furthermore, the specific binding of [(125)I] α-bungarotoxin was not attenuated by bisabolol. Choline-induced currents in CA1 interneurons of rat hippocampal slices were also inhibited with IC50 of 4.6 µM. Collectively, our results suggest that bisabolol directly inhibits α7-nAChRs via a binding site on the receptor channel.

Control of porcine lacrimal gland secretion by non-cholinergic, non-adrenergic nerves: effects of electrical field stimulation, VIP and NPY.

This study employs the technique of electrical field stimulation (EFS) to characterise the effects of endogenous neurotransmitters on protein secretion in the in vitro pig lacrimal gland. The effects of exogenous applications of neurotransmitters on protein output and peroxidase secretion were also investigated for comparative purposes. EFS evoked frequency-dependent (5-20 Hz) increases in protein secretion. The EFS-evoked protein output was abolished with the nerve blocking drug tetrodotoxin (10(-6) M, TTX). Elevated potassium (100 mM KCl) can stimulate protein output in the presence of TTX. Exogenous application of either acetylcholine (ACh, 10(-9)-10(-4) M) or noradrenaline (NA, 10(-8)-10(-4) M) can also result in protein secretion, but they have no detectable effect on peroxidase secretion. In the presence of the cholinergic antagonist, atropine (10(-5) M) the EFS-induced protein output was reduced but not abolished. This atropine-resistant and non-cholinergic nerve-mediated component was further reduced in the combined presence of atropine, phentolamine, and propranolol (all 10(-5) M). When vasoactive intestinal polypeptide (VIP) receptor antagonist (10(-6) M [4-Cl-D-Phe6-Leu17]-VIP) was combined with the cholinergic and adrenergic antagonists, EFS caused a small but detectable increase in protein output. Exogenous application of either 10(-9) M VIP or 10(-9) M neuropeptide-Y (NPY) resulted in protein secretion. Combination of both VIP and NPY only induced an additive effect on protein output. Theophylline (10(-4) M), a phosphodiesterase inhibitor, evoked a small increase in protein output and had no significant effect on the secretory responses elicited by either VIP or NPY. In contrast, theophylline potentiated the non-cholinergic, non-adrenergic EFS-induced protein secretion. The results indicate that protein secretion from the porcine lacrimal gland may be controlled by cholinergic, adrenergic and non-cholinergic, non-adrenergic nerves. The peptidergic neurotransmitters may be VIP and other related neuropeptide(s). In addition to these neurophysiological studies, our results confirm previous findings that the porcine lacrimal nerves contain abundant quantity of NPY and VIP.

Chronic effects of streptozotocin-induced diabetes on the ultrastructure of rat ventricular and papillary muscle.

Contractile dysfunctions have been demonstrated in different experimental models of diabetes which have similar characteristics to many of the abnormalities found in the clinical setting. Administration of streptozotocin (STZ) to young adult rats induces beta-cell necrosis of the pancreas which gives rise to hypoinsulinaemia and hyperglycaemia, features which are also seen in untreated type 1 clinical diabetes. We have investigated the chronic effects of STZ-induced diabetes on contraction in rat ventricular myocytes and ultrastructure of cardiac muscle. Diabetes was induced in male Wistar rats (230-270 g) with a single injection of STZ (60 mg kg(-1)). At 2 and 10 months after STZ treatment, the amplitude of contraction was larger in diabetic compared to control myocytes. Time to peak contraction was significantly longer at 2 months but appeared to normalise at 10 months after STZ treatment. In contrast, time to half relaxation of contraction was not significantly different after 2 months but was significantly reduced at 10 months after STZ treatment compared to control. Transmission electron microscope examination of cardiac muscle showed that the ultrastructure of cardiac muscle, especially structures associated with contraction, were not greatly altered after STZ treatment. Sarcomere lengths were not significantly different in papillary or ventricular muscle at 4 or 8 months after STZ treatment compared to control. Our data provide evidence that morphological defects in contractile myofilaments and associated structures cannot explain contractile dysfunctions seen in ventricular myocytes from STZ-treated animals.

Characterisation of ventricular myocyte shortening after administration of streptozotocin (STZ) to neonatal rats.

Human and animal studies have shown that diabetes mellitus can be associated with altered cardiac function that is independent of vascular complications. Streptozotocin (STZ) is a diabetogenic agent and when administered to rats causes selective beta-cell necrosis which is accompanied by a drastic reduction in plasma insulin and hyperglycaemia. We have investigated the characteristics of shortening in ventricular myocytes isolated from rat heart at 10 months after administration of STZ to neonatal rats at 2 days of age. The characteristics of shortening in myocytes from the STZ-treated neonatal rat compared to shortening in myocytes from the STZ-treated young adult rat are discussed. STZ-treated rats gained significantly less weight compared to age-matched controls. Although non-fasting blood glucose was not significantly different in STZ-treated rats they were found to be markedly glucose intolerant when given an intraperitoneal challenge of glucose (2 g/kg) after an overnight fast. During electrical stimulation (1 or 2 Hz) ventricular myocyte resting length, time to peak shortening, time to half relaxation and amplitude of shortening were not altered after STZ treatment. The imposition of rest periods (2-60 s) after trains of electrically stimulated (1 Hz) steady-state contractions resulted in a potentiation of the contraction, which immediately followed the rest period (post rest potentiation). Post rest potentiation was larger, following rest periods between 20 and 60 s, in myocytes from STZ-treated rats compared to controls. The absence of major alterations to the amplitude and kinetics of contraction in myocytes from STZ-treated neonatal rats at 10 months after treatment might be explained by a partial recovery of the beta-cells in the growing animal.

Catecholamines in the heart and adrenal gland of the STZ-diabetic rat.

The concentrations of noradrenaline (NA), adrenaline (ADR), 5-hydroxyindoleacetic acid (5-HIAA), serotonin (5-HT) and dopamine (DOP) have been studied in the left ventricle and the left adrenal gland of control and streptozotocin (STZ) - treated rats at various intervals (12, 24, 30, 34, 38 and 42 weeks) after the induction of diabetes. The only amines detected in the heart were NA, 5-HIAA and DOP, whereas those detected in the adrenal gland were NA and ADR. Differential changes in the catecholamine concentrations occurred in the heart and the adrenal gland at different stages of the metabolic disorder. In the heart the initial changes in short-term diabetes included an increase in NA concentration but this did not persist in the longer term diabetic animals (30-38 weeks following STZ injection). In the adrenal gland there was an initial reduction followed by a steady increase in the concentration of NA and ADR throughout the period of the study.

Effects of extracellular magnesium and beta adrenergic stimulation on contractile force and magnesium mobilization in the isolated rat heart.

This study investigates the metabolism of the divalent cation, magnesium (Mg2+) in the isolated perfused Langendorff's rat heart and ventricular slices in the absence and presence of catecholamines including isoprenaline, noradrenaline and adrenaline. Perfusion of the isolated rat heart with a physiological salt solution containing elevated extracellular Mg2+ [Mg2+]o (2.4 mM-6.0 mM) resulted in a marked and progressive decrease in the amplitude of contraction compared to control [Mg2+]o (1.2 mM). In contrast, perfusion of hearts with low (0-0.6 mM) [Mg2+]o caused a small transient increase in the amplitude of contraction which was often accompanied by arrhythmic activity. Perfusion of the heart with a nominally Mg2+ free medium resulted in a time-dependent net efflux of Mg2+ reaching a steady state after approximately 40-50 min of perfusion. This release of Mg2+ was associated with a concurrent decrease in total heart Mg2+. Stimulation of the heart with the beta adrenergic agonist, isoprenaline (10(-7) M) caused large increases in net Mg2+ efflux which was associated with marked increased in both rate and the amplitude of contraction. Similar effects on Mg2+ efflux were also observed during perfusion of the heart with the adenylate cyclase activator, forskolin (10(-5) M). Superfusion of paced ventricular segments with either isoprenaline, adrenaline or noradrenaline (all 10(-6) M) also resulted in a marked transient net efflux of Mg2+. Pre-treatment of segments with the beta adrenergic antagonist, propranolol (10(-5) M) competitively blocked the Mg2+ efflux evoked by the catecholamines. Similarly, pre-treatment of segments with the calcium (Ca2+) channel blocker, verapamil (10(-5) M) caused a significant (P < 0.05) decrease in net Mg2+ efflux evoked by isoprenaline. The results of this study indicate that (1) the perturbation of [Mg2+]o has an important influence on myocardial contractility and (2) the mobilization of Mg2+ in the heart is associated with beta adrenergic stimulation possibly via an elevation in intracellular adenosine 3.5 cyclic monophosphate (cyclic AMP).