A study of the electrical basis for the inhibition and excitation in autonomically innervated smooth muscle

The aim of this thesis was to investigate the electrical and mechanical responses to inhibitory non-adrenergic noncholinergic (NANC) nerve stimulation in the bovine retractor penis muscle (BRP) and compare them with those to an inhibitory extract made from this muscle. The extract may contain the NANC inhibitory transmitter of the BRP and possibly of other smooth muscles. Because of species differences in the electrical response to NANC nerves in the rat and rabbit anococcygeus the effects of the extract on these tissues was also investigated. Prior to the investigation of the extract, both the excitatory and inhibitory responses to field stimulation in the BRP, and the effects of passive membrane potential displacement were studied using conventional intra- or extracellular (sucrose gap) recording techniques. The majority of cells in the BRP were electrically quiescent independent of the resting tone. The most frequent (in approximately 25% of preparations) form of spontaneous activity, oscillations in membrane potential and tone, may represent a pacemaker activity. The BRP had cable properties; the time constant and space constant indicated a high membrane resistance. In the absence of tone, field stimulation of the BRP evoked excitatory junction potentials (ejps) in every cell impaled and contractions, graded with the strength, frequency and number of pulses; spikes were not observed. Guanethidine (1-3 x 10-5M) abolished the ejps and contractions, confirming their adrenergic origin. Noradrenaline added exogenously depolarised and contracted the muscle. These effects were blocked by the a-adrenoceptor antagonists, phentolamine and prazosin. However, phentolamine (2.5x 10-6M) inhibited the contraction without reducing the ejp significantly. These effects may be independent of adrenoceptor blockade or the ejp may be mediated by a substance other than noradrenaline (e.g. ATP) released from adrenergic nerves. Prazosin (1.4 x lO-6M) failed to block either the ejp or contraction, indicating the possible existence of two types of adrenoceptor in the BRP; one activated by neuronally-released and the other by exogenously-added noradrenaline. ATP, a contaminant in the extract, also depolarised and contracted the BRP. Physostigmine reduced whilst atropine enhanced the ejps and contractions without similarly affecting the response to exogenous noradrenaline. This confirmed the presence of a cholinergic inhibitory innervation acting on the excitatory adrenergic fibres (Klinge and Sjostrand, 1977). TEA (1 x lO-4M) enhanced the ejp and contraction. Higher concentrations (0.5 to 10 x 10-3M) depolarised, increased the tone and evoked electrical and mechanical oscillations but no spikes. The depolarisation and contraction to exogenous noradrenaline were not enhanced, indicating that TEA acts on the adrenergic nerves. Some post-synaptic effect to block K+ channels also seems likely. The relationship between ejp amplitude and membrane potential in the double sucrose gap was linear and indicated a reversal potential more positive than -30mV. Electrotonic pulse amplitude decreased during the ejp, indicating an increased membrane conductance. Ejps and contractions were reduced following the replacement of the NaCl of the Krebs solution with sodium glutamate. This may be due to the effects of glutamate itself (e.g. Ca2+ chelation) rather than reduction in the membrane Cl- gradient. Tone usually developed spontaneously and was accompanied by membrane depolarisation (from -53 to -45mV) which may open voltage-dependent channels, causing Ca2+ entry and/or its release from intracellular binding sites. Field stimulation produced inhibitory potentials (ijps) and relaxations graded with the strength and number of pulses but showing little frequency dependence. Rebound depolarisation and contraction often followed the ijp and relaxation. Tetrodotoxin (3 x IO-6M), but not adrenergic or cholinergic antagonists, abolished the ijp and relaxation, confirming their non-adrenergic non-cholinergic neurogenic nature. The extract, prepared and acid-activated as described by Gillespie, Hunter and Martin (1981), hyperpolarised and relaxed the BRP, as did sodium nitroprusside and adenosine triphosphate (ATP). Unlike the activated extract or sodium nitroprusside, desensitisation to ATP occurred rapidly and without any change in the inhibitory electrical or mechanical responses to field stimulation. The ijp and relaxation in the BRP were insensitive to apamin but abolished by oxyhaemoglobin (4-8 x 10-6M), as were the responses to extract and sodium nitroprusside. In TEA (10-2M), field stimulation evoked relaxations with no accompanying electrical change. The ijp may be unconnected with or additional to another mechanism producing relaxation. The relationship between membrane potential and ijp in the BRP was non-linear. Ijp amplitude was initially increased during membrane potential displacement from -45mV to approximately -60mV. Thereafter (-60 to -l03mV) the ijp was reduced. Ijps were abolished at -27 and -103mV; reversal was not observed. The hyperpolarisation to extract was also enhanced during passive displacement of the membrane potential to more negative values (-57mV). Membrane resistance increased during the ijp. The extract produced inconsistent changes in membrane resistance, possibly because of the presence of more than one active component. K+ withdrawal failed to enhance the ijp or hyperpolarisation to extract and 20mM K+ did not abolish the the ijp at membrane potentials exceeding EK (-49mV). Thus, the ijp or hyperpolarisation to extract are unlikely to be mediated by an increased K+ conductance. Reducing the Cl- abolished the hyperpolarisation to field stimulation and extract. This occurred more quickly than the anticipated reduction in the Cl- gradient and may be due to Ca2+ chelation by the anion substitute (glutamate or benzenesulphonate) or blockade of the resting conductance which is normally inactivated by the transmitter. Ouabain (1-5x 10-5M), which reduces both the Na+ and Cl- gradients, abolished the ijp, implicating either of these ions as the ionic species involved. In the rat and rabbit anococcygeus, field stimulation and extract each reduced guanethidine-induced tone. This was unaccompanied in the majority of cells in the rat by any significant electrical response. In the remaining cells, inhibition of the membrane potential oscillations occurred. The rabbit anococcygeus differed in that inhibition of the electrical oscillations was observed in every cell exhibiting this behaviour. However, the majority of cells in the rabbit were electrically quiescent and showed only small hyperpolarisations to field stimulation and no electrical response to extract. Apamin (1 x 10-7M) failed to block the electrical and mechanical response to field stimulation in the rabbit but did inhibit transiently that to extract. The latter effect may be due to the initial excitatory effects of apamin. The similarities between the electrical effects of the extract and those of inhibitory nerve stimulation in the BRP, rat and rabbit anococcygeus muscles are generally consistent with their being mediated by the same active component. Moreover, the ijp in the BRP shows properties which have not been reported in other non-adrenergic noncholinergically innervated smooth muscles.

The regulation of AMP-activated protein kinase by vascular endothelial growth factor

The function of the vascular endothelium is to maintain vascular homeostasis, by providing an anti-thrombotic, anti-inflammatory and vasodilatory interface between circulating blood and the vessel wall, meanwhile facilitating the selective passage of blood components such as signaling molecules and immune cells. Dysfunction of the vascular endothelium is implicated in a number of pathological states including atherosclerosis and hypertension, and is thought to precede atherogenesis by a number of years. Vascular endothelial growth factor A (VEGF) is a crucial mitogenic signaling molecule, not only essential for embryonic development, but also in the adult for regulating both physiological and pathological angiogenesis. Previous studies by our laboratory have demonstrated that VEGF-A activates AMP-activated protein kinase (AMPK), the downstream component of a signaling cascade important in the regulation of whole body and cellular energy status. Furthermore, studies in our laboratory have indicated that AMPK is essential for VEGF-A-stimulated vascular endothelial cell proliferation. AMPK activation typically stimulates anabolic processes and inhibits catabolic processes including cell proliferation, with the ultimate aim of redressing energy imbalance, and as such is an attractive therapeutic target for the treatment of obesity, metabolic syndromes, and type 2 diabetes. Metabolic diseases are associated with adverse cardiovascular outcomes and AMPK activation is reported to have beneficial effects on the vascular endothelium. The mechanism by which VEGF-A stimulates AMPK, and the functional consequences of VEGF-A-stimulated AMPK activation remain uncertain. The present study therefore aimed to identify the specific mechanism(s) by which VEGF-A regulates the activity of AMPK in endothelial cells, and how this might differ from the activation of AMPK by other agents. Furthermore, the role of AMPK in the pro-proliferative actions of VEGF-A was further examined. Human aortic and umbilical vein endothelial cells were therefore used as a model system to characterise the specific effect(s) of VEGF-A stimulation on AMPK activation. The present study reports that AMPK α1 containing AMPK complexes account for the vast majority of both basal and VEGF-A-stimulated AMPK activity. Furthermore, AMPK α1 is localized to the endoplasmic reticulum when sub-confluent, but translocated to the Golgi apparatus when cells are cultured to confluence. AMPK α2 appears to be associated with a structural cellular component, but neither α1 nor α2 complexes appear to translocate in response to VEGF-A stimulation. The present study confirms previous reports that when measured using the MTS cell proliferation assay, AMPK is required for VEGF-A-stimulated endothelial cell proliferation. However, parallel experiments measuring cell proliferation using the Real-Time Cell Analyzer xCELLigence system, do not agree with these previous reports, suggesting that AMPK may in fact be required for an aspect of mitochondrial metabolism which is enhanced by VEGF-A. Studies into the mitochondrial activity of endothelial cells have proved inconclusive at this time, but further studies into this are warranted. During previous studies in our laboratory, it was suggested that VEGF-A-stimulated AMPK activation may be mediated via the diacylglycerol (DAG)-sensitive transient receptor potential cation channel (TRPCs -3, -6 or -7) family of ion channels. The present study can neither confirm, nor exclude the expression of TRPCs in vascular endothelial cells, nor rule out their involvement in VEGF-A-stimulated AMPK activation; more specific investigative tools are required in order to characterise their involvement. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP)-stimulated Ca2+ release from acidic intracellular organelles is not required for AMPK activation by VEGF-A. Despite what is known about the mechanisms by which AMPK is activated, far less is known concerning the downregulation of AMPK activity, as observed in human and animal models of metabolic disease. Phosphorylation of AMPK α1 Ser485 (α2 Ser491) has recently been characterised as a mechanism by which the activity of AMPK is negatively regulated. We report here for the first time that VEGF-A stimulates AMPK α1 Ser485 phosphorylation independently of the previously reported AMPK α1 Ser485 kinases Akt (protein kinase B) and ERK1/2 (extracellular signal-regulated kinase 1/2). Furthermore, inhibition of protein kinase C (PKC), the activity of which is reported to be elevated in metabolic disease, attenuates VEGF-A- and phorbol 12-myristate 13-acetate (PMA)-stimulated AMPK α1 Ser485 phosphorylation, and increases basal AMPK activity. In contrast to this, PKC activation reduces AMPK activity in human vascular endothelial cells. Attempts to identify the PKC isoform responsible for inhibiting AMPK activity suggest that it is one (or more) of the Ca2+-regulated DAG-sensitive isoforms of PKC, however cross regulation of PKC isoform expression has limited the present study. Furthermore, AMPK α1 Ser485 phosphorylation was inversely correlated with human muscle insulin sensitivity. As such, enhanced AMPK α1 Ser485 phosphorylation, potentially mediated by increased PKC activation may help explain some of the reduced AMPK activity observed in metabolic disease.