Rania Abu-Hamdah

Structure and Composition of the Fusion Pore

Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. Fusion pores were stable structures at the cell plasma membrane where secretory vesicles dock and fuse to release vesicular contents. In the present study, transmission electron microscopy confirms the presence of fusion pores and reveals their detailed structure and association with membrane-bound secretory vesicles in pancreatic acinar cells. Immunochemical studies demonstrated that t-SNAREs, NSF, actin, vimentin, alpha-fodrin and the calcium channels alpha1c and beta3 are associated with the fusion complex. The localization and possible arrangement of SNAREs at the fusion pore are further demonstrated from combined AFM, immunoAFM, and electrophysiological measurements. These studies reveal the fusion pore or porosome to be a cup-shaped lipoprotein structure, the base of which has t-SNAREs and allows for docking and release of secretory products from membrane-bound vesicles.

Vesicle swelling regulates content expulsion during secretion

The involvement of secretory vesicle swelling has been proposed in secretion; however, little is known about its role. Using both the pancreatic acinar cell and neuronal model, we show secretory vesicle swelling in live cells. Our study reveals that vesicle swelling potentiates its fusion at the cell plasma membrane, and is required for expulsion of intravesicular contents. Since the extent of swelling is directly proportional to the amount of vesicular contents expelled, this provides cells with the ability to regulate release of secretory products. These direct observations of the requirement of secretory vesicle swelling in secretion, provides an understanding of the appearance of partially empty vesicles following the process.

Regulation of the water channel aquaporin-1: isolation and reconstitution of the regulatory complex

Aquaporins (AQP) are involved in rapid and active gating of water across biological membranes. The molecular regulation of AQP is unknown. Here we report the isolation, identification and reconstitution of the regulatory complex of AQP‐1. AQP‐1 and Gαi3have been implicated in GTP‐induced gating of water in zymogen granules (ZG), the secretory vesicles in exocrine pancreas. In the present study, detergent‐solubilized ZGs immunoprecipitated with monoclonal AQP‐1 antibody, co‐isolates AQP‐1, PLA2, Gαi3, potassium channel IRK‐8, and the chloride channel ClC‐2. Exposure of ZGs to either the potassium channel blocker glyburide, or the PLA2 inhibitor ONO‐RS‐082, blocked GTP‐induced ZG swelling. RBC known to possess AQP‐1 at the plasma membrane, swell on exposure to the Gαi—agonist mastoparan, and respond similarly to ONO‐RS‐082 and glyburide, as ZGs. Liposomes reconstituted with the AQP‐1 immunoisolated complex from solubilized ZG, also swell in response to GTP. Glyburide or ONO‐RS‐082 abolished the GTP effect. Immunoisolate‐reconstituted planar lipid bilayers demonstrate conductance, which is sensitive to glyburide and an AQP‐1 specific antibody. Our results demonstrate a Gαi3‐PLA2 mediated pathway and potassium channel involvement in AQP‐1 regulation.

Patch Clamped Single Pancreatic Zymogen Granules: Direct Measurements of Ion Channel Activities at the Granule Membrane

Background/Aim: Pancreatic acinar cells are involved in the secretion of digestive enzymes. Digestive enzymes in pancreatic acinar cells are stored in membrane-bound secretory vesicles called zymogen granules (ZGs). The swelling of ZGs is implicated in the regulation of the expulsion of intravesicular contents during secretion. The molecular mechanism of ZG swelling has been previously elucidated. It has been further demonstrated that the water channel aquaporin-1, the potassium channel IRK-8, and the chloride channel CLC-2, are present in the ZG membrane and involved in ZG swelling. However, a direct measurement of these ion channels at the ZG membrane in intact ZGs had not been performed. The aim of this study was to investigate the electrical activity of single ZGs and verify the types of channels found within their membrane. Methods: ZGs from pancreatic acinar cells were isolated from the pancreas of Sprague-Dawley rats. Direct measurements of whole vesicle currents, in the presence and absence of ion channel blockers (quinine, glyburide and DIDS), were recorded following successful patching of single ZGs. Conclusion: In this study, we were able, for the first time, to patch single ZGs and study ion channels in their membrane. We were able to record currents across the ZG membrane and, utilizing ion channel blockers, confirm the presence of the chloride channels CLC-2 and the potassium channel IRK-8 (Kir6.1), and additionally demonstrate the presence of a second chloride channel CLC-3.

Involvement of vH+-ATPase in synaptic vesicle swelling

Secretory vesicle swelling is central to cell secretion, but the underlying mechanism of vesicle swelling, particularly synaptic vesicles, is not completely understood. The Gαi3‐PLA2‐mediated involvement of water channel AQP‐1 in the regulation of secretory vesicle swelling in exocrine pancreas and the Gαo‐mediated AQP‐6 involvement in synaptic vesicle swelling in neurons have previously been reported. Furthermore, the role of vH+‐ATPase in neurotransmitter transport into synaptic vesicles has also been shown. Using nanometer‐scale precision measurements of isolated synaptic vesicles, the present study reports for the first time the involvement of vH+‐ATPase in GTP‐Gαo‐mediated synaptic vesicle swelling. Results from this study demonstrate that the GTP‐Gαo‐mediated vesicle swelling is vH+‐ATPase dependent and pH sensitive. Zeta potential measurements of isolated synaptic vesicles further demonstrate a bafilomycin‐sensitive vesicle acidification, following the GTP‐Gαo‐induced swelling stimulus. Water channels are bidirectional and the vH+‐ATPase inhibitor bafilomycin decreases both the volume of isolated synaptic vesicles and GTP‐mastoparan stimulated swelling, suggesting that vH+‐ATPase is upstream of AQP‐6, in the pathway leading from Gαo‐stimulated swelling of synaptic vesicles. Vesicle acidification is therefore a prerequisite for AQP‐6‐mediated gating of water into synaptic vesicles.

Attenuated muscle metaboreflex-induced increases in cardiac function in hypertension.

Sympathoactivation may be excessive during exercise in subjects with hypertension, leading to increased susceptibility to adverse cardiovascular events, including arrhythmias, infarction, stroke, and sudden cardiac death. The muscle metaboreflex is a powerful cardiovascular reflex capable of eliciting marked increases in sympathetic activity during exercise. We used conscious, chronically instrumented dogs trained to run on a motor-driven treadmill to investigate the effects of hypertension on the mechanisms of the muscle metaboreflex. Experiments were performed before and 30.9 ± 4.2 days after induction of hypertension, which was induced via partial, unilateral renal artery occlusion. After induction of hypertension, resting mean arterial pressure was significantly elevated from 98.2 ± 2.6 to 141.9 ± 7.4 mmHg. The hypertension was caused by elevated total peripheral resistance. Although cardiac output was not significantly different at rest or during exercise after induction of hypertension, the rise in cardiac output with muscle metaboreflex activation was significantly reduced in hypertension. Metaboreflex-induced increases in left ventricular function were also depressed. These attenuated cardiac responses caused a smaller metaboreflex-induced rise in mean arterial pressure. We conclude that the ability of the muscle metaboreflex to elicit increases in cardiac function is impaired in hypertension, which may contribute to exercise intolerance.

Exaggerated coronary vasoconstriction limits muscle metaboreflex-induced increases in ventricular performance in hypertension

Increases in myocardial oxygen consumption during exercise mainly occur via increases in coronary blood flow (CBF) as cardiac oxygen extraction is high even at rest. However, sympathetic coronary constrictor tone can limit increases in CBF. Increased sympathetic nerve activity (SNA) during exercise likely occurs via the action of and interaction among activation of skeletal muscle afferents, central command, and resetting of the arterial baroreflex. As SNA is heightened even at rest in subjects with hypertension (HTN), we tested whether HTN causes exaggerated coronary vasoconstriction in canines during mild treadmill exercise with muscle metaboreflex activation (MMA; elicited by reducing hindlimb blood flow by ~60%) thereby limiting increases in CBF and ventricular performance. Experiments were repeated after α1-adrenergic blockade (prazosin; 75 µg/kg) and in the same animals following induction of HTN (modified Goldblatt 2K1C model). HTN increased mean arterial pressure from 97.1 ± 2.6 to 132.1 ± 5.6 mmHg at rest and MMA-induced increases in CBF, left ventricular dP/dtmax, and cardiac output were markedly reduced to only 32 ± 13, 26 ± 11, and 28 ± 12% of the changes observed in control. In HTN, α1-adrenergic blockade restored the coronary vasodilation and increased in ventricular function to the levels observed when normotensive. We conclude that exaggerated MMA-induced increases in SNA functionally vasoconstrict the coronary vasculature impairing increases in CBF, which limits oxygen delivery and ventricular performance in HTN. NEW & NOTEWORTHY We found that metaboreflex-induced increases in coronary blood flow and ventricular contractility are attenuated in hypertension. α1-Adrenergic blockade restored these parameters toward normal levels. These findings indicate that the primary mechanism mediating impaired metaboreflex-induced increases in ventricular function in hypertension is accentuated coronary vasoconstriction.

Addendum to “Regulation of the water channel aquaporin‐1: isolation and reconstitution of the regulatory complex” [Cell Biol. Int. 28(1) (2004) 7–17]*

In a routine assay using atomic force microscopy (AFM) to image red blood cells (RBCs) under various experimental conditions, rapid increase in RBC size following exposure to water (up to a 21% increase in volume), or mastoparan (up to a 73% increase in volume) was demonstrated. The increase in size of RBCs following exposure to water was much lower when assessed by the AFM; however, this modest increase in RBC volume following exposure to water was undetectable by our light-scattering analysis. Although, 150% increase in light-scattering intensity was observed when RBCs were exposed to mastoparan (Fig. 5B; AbuHamdah et al., 2004), little change in light-scattering was detected following exposure to water. Similar to the increase in light-scattering following exposure to mastoparan, we were able to detect an increase in lightscattering when RBCs were exposed to tributyltin chloride (TBT), as previously reported by Ohkuma