Paula Grammas

Identification of muscarinic receptors in rat cerebral cortical microvessels.

Abstract: Microvessels isolated from rat cerebral cortex consist mainly of capillaries (>85%). Fresh, intact microvessel preparations have been analyzed by radioligand binding techniques for muscarinic receptors. Scatchard analysis of specific quinuclidinyl benzilate (QNB) binding indicates that microvessels possess a large number of muscarinic sites (914 fmol/mg protein) of high affinity (Kd= 0.034 nM). The association and dissociation rate constants (0.37 min−1 nM−1 and 0.0067 min−1, respectively) yield an equilibrium Kd of 0.018 nM. Displacement of [3H]QNB by muscarinic ligands and control substances is typical of muscarinic receptors. The results indicate that cerebral microvessels possess a large population of muscarinic receptors.

ISolation and characterization of cerebral resistance vessel endothelium in culture

Organ-derived endothelia have been shown to exhibit distinct patterns of morphology and growth responsiveness in vitro. This report describes the development, cloning and establishment of long-term serial cultures of rat vascular endothelial cells derived from cerebrocortical resistance vessels (small arteries and arterioles). Modification of our previous published technique for establishing resistance vessel-derived smooth muscle cells (RV-SMC) resulted in enhanced levels of endothelial outgrowth from collagenase-treated microvessel fragments. Although primary culture growth consisted predominantly of SMC, subsequent subcultivation of these cultures revealed the presence of distinct endothelial cell clusters within the SMC monolayer. Serial cloning of these isolates resulted in a homogeneous population of cells with the characteristic endothelial cobblestone growth pattern and positive immunofluorescence for factor VIII-related antigen. Previously established RV-SMC frozen stocks provided an additional source for obtaining resistance vessel endothelial cells. This was made possible by the slow proliferation rate of early-passage RV-SMC and their inability to withstand freezing procedures. Endothelial cells from both preparations were identical and designated resistance vessel derived endothelial cells RV-EC. Upon long-term cultivation (> P15), confluent RV-EC cultures expressed spontaneous multicellular cord development that stained positive for factor VIII-related antigen. Cell growth studies demonstrated that RV-EC were capable of significant growth when maintained in serum-free conditions. Growth kinetics using serum-free conditioned medium demonstrated mitogenic activity indicating the presence of an autocrine growth factor. Increase growth responsiveness was also noted in RV-EC when treated with a variety of peptide growth factors. These results indicate that resistance vessel endothelium can be successfully isolated and maintained in long-term serial cultures. Furthermore, the availability of cultured EC and SMC from this unique microvascular site will enable examination of cerebrovascular endothelial-smooth muscle cell interactions in vitro and may help to elucidate the mechanisms of altered vascular function in disease states.

Cerebrovascular angiotensin II receptors in spontaneously hypertensive rats

The objective of this study was to characterize angiotensin II (AII) receptors in cerebral capillary endothelium and to examine whether the first step in All responsiveness, namely All receptor binding, is aberrant in cerebral microvessels obtained from adult spontaneously hypertensive rats (SHR). The binding of [3H]angiotensin II to isolated cerebrocortical microvessels from Sprague-Dawley, Wistar-Kyoto, and SHR rats was used to characterize All receptors on these vessels. Kinetic experiments yielded an equilibrium-derived Kd (dissociation rate constant/association rate constant) very close to that obtained from Scatchard analysis of saturation binding data. The data indicated that the two normotensive control strains exhibited comparable All receptor affinity and binding capacity. In contrast, experiments with microvessels from adult SHR indicated a significantly higher sbiBmax for All receptors relative to controls. Although experiments assessing functional endothelial alterations in the SHR to All remain to be performed, the increase in All receptor number suggests that an abnormality in vascular AII responsiveness may play an important role in this model of hypertension.

Control of [3H]ouabain binding to cerebromicrovascular (Na+ + K+)-ATPase by metal ions and proteins

The (Na+ + K+)-ATPase is localized to the cerebral endothelium, i.e. the blood-brain barrier, and is important for the maintenance of the brain electrolyte environment. Data from the present study indicate that Pb2+ inhibits the binding of [3H]ouabain to the cerebral microvascular (Na+ + K+)-ATPase in a time- and dose-dependent manner. Pb2(+)-induced inhibition developed slowly with a maximum obtained after 40 min. Inhibition of [3H]ouabain binding to the enzyme was 48% at 10 microM Pb2+ and appeared maximal (89%) at 100 microM Pb2+ when compared to [3H]ouabain binding in untreated microvessels at 40 min. In contrast, 100 microM Al3+ caused a 55% increase in [3H]ouabain binding to the (Na+ + K+)-ATPase, relative to untreated microvessels at 40 min. Insulin or bovine serum albumin stimulated [3H]ouabain binding to the enzyme when added at similar concentrations. However, the addition of both insulin and bovine serum albumin did not result in an additive effect. These results show that insulin exerts a nonspecific effect on [3H]ouabain binding to the (Na+ + K+)-ATPase similar to that evoked by bovine serum albumin. However, the metal ions Pb2+ and Al3+ provoke selective alterations in the cerebromicrovascular (Na+ + K+)-ATPase with Pb2+ inhibiting and Al3+ stimulating [3H]ouabain binding.

Angiotensin II and atrial natriuretic factor receptor interactions at the blood-brain barrier

Data from several laboratories indicate that cerebral endothelial cells possess cell surface receptors for numerous vasoactive agents including angiotensin II (AII) and atrial natriuretic factor (ANF). The intracellular messengers of these receptors as well as possible receptor interactions were explored. ANF increased cGMP 10-fold over basal levels while incubation of the microvessels with AII did not significantly affect the level of this nucleotide. In contrast, AII significantly potentiated the increase in cGMP by ANF. Incubation of cerebral microvessels with AII resulted in a significant increase in the intracellular mediator of PI hydrolysis, 1,2-diacylglycerol (DG). ANF had no affect on DG or on the AII mediated increase of DG. Finally, data at the level of receptor binding indicated that while ANF decreased [3H]angiotensin binding to cerebral microvessels, AII had no effect on the binding of ANF to its receptor. The results of the present study demonstrate that AII can potentiate the regulation of cGMP by ANF and suggest the possibility of receptor interactions in control of blood-brain barrier function.

Control of the Na+,K(+)-ATPase under normal and pathological conditions.

The Na+,K(+)-ATPase is an important enzyme in determining the ionic milieu of the cerebromicrovasculature and neurons. The effect of hypertension or aging on this enzyme, as well as its susceptibility to regulation by fatty acids or aluminum, is the focus of this study. A significant increase (34%) in the apparent affinity constant (KD) but no change in the maximum binding capacity (Bmax) for [3H]ouabain binding to the cerebromicrovascular Na+,K(+)-ATPase occurs after induction of acute hypertension. In addition, long chain unsaturated fatty acids stimulate the binding of [3H]ouabain to the enzyme in microvessels from normotensive and hypertensive rats. The synaptosomal Na+,K(+)-ATPase is sensitive to aluminum. AlCl3 (1-100 microM) inhibits the K(+)-dependent-p-nitrophenylphosphatase (K(+)-NPPase) activity of the Na+,K(+)-ATPase in a dose-dependent manner. AlCl3 (100 microM) decreases the Vmax by 14% but does not alter the KM, suggestive of non-competitive inhibition. The enzyme from aged brain displays a greater Vmax, but shows the same susceptibility to AlCl3 as the enzyme from younger brain. In summary, disruption of the Na+,K(+)-ATPase may underlie, at least in part, abnormalities of nerve and vascular cell function in disorders where elevated concentrations of fatty acids or metal ions are involved.

Cholinergic‐Adrenergic Receptor Interactions in Cerebral Microvessels

Enriched capillary preparations isolated from rat cerebral cortex were used to evaluate cholinergic‐adrenergic receptor interactions in cerebral endothelium. Possible receptor interactions were determined by measuring an intracellular mediator, cyclic AMP and alterations in GTP‐sensitive agonist binding. Unstimulated microvessel homogenates generate 66 ± 16 pmol/mg/10 min of cyclic AMP. Adrenergic agonists norepinephrine and isoproterenol increase cyclic AMP to 147 ± 31 and 149 ± 23 pmol/mg/10 min, respectively. Addition of the muscarinic agonist carbachol has no effect on basal cyclic AMP but it completely blocks the stimulation elicited by adrenergic agonists. The displacement of quinuclidinyl benzilate (QNB) by carbachol yields an IC50 of 1.5 ± 0.45 x 10−4M and a Hill coefficient of 0.54 ± 0.07, indicating a heterogeneous population of binding sites. Guanine nucleotides shift the displacement curve to the right (IC50, 4.7 ± 0.16 x 10−4M) and convert the binding site population to greater homogeneity (0.76 ± 0.18). Isoproterenol prevents both the affinity shift and binding site conversion evoked by guanine nucleotides. These data suggest that cholinergic‐adrenergic interactions occur at both the level of receptor binding and the generation of an intracellular messenger. Since cyclic AMP has been purported to play a role in regulation of blood–brain barrier permeability, the existence of adrenergic‐cholinergic, i.e., excitatory‐inhibitory modulators of adenylate cyclase in cerebral endothelium, suggests that these receptors may mediate physiological and/or pathological alterations of cerebrovascular permeability.

Effect of Fatty Acids on [3H]Ouabain Binding to Cerebromicrovascular (Na++ K+)-ATPase

Abstract: The effects of short‐ and long‐chain fatty acids on the cerebromicrovascular (Na++ K+)‐ATPase were investigated using specific [3H]ouabain binding to the enzyme. Specific binding increased linearly with total microvessel protein (37–110 μg) and was time‐dependent with maximum binding obtained by 10 min. Arachidonic acid, but not palmitic acid, stimulated [3H]ouabain binding in a dose‐dependent manner, with a 105% increase over basal levels at 100 μM arachidonic acid. Preincubation of the microvessels with arachidonic acid did not alter the stimulation observed. 4‐Pentenoic acid stimulated [3H]ouabain binding only at high concentrations (10 mM). Scatchard analysis of [3H]ouabain binding to untreated microvessels yielded a single class of “high‐affinity” binding sites with an apparent binding affinity (KD) of 64.7 ± 2.0 nM and a binding capacity (Bmax) of 10.1 ± 1.5 pmol/mg protein. In the presence of 100 μM arachidonic acid, a monophasic Scatchard plot also was obtained, but the KD significantly decreased to 51.9 ± 2.7 nM (p < 0.01), whereas the Bmax remained virtually unchanged (12.5 ± 1.2 pmol/mg protein). The stimulation of [3H]ouabain binding in the presence of arachidonic acid was potentiated by 4‐pentenoic acid, but not by indomethacin or eicosatetraynoic acid. These data suggest that long‐chain polyunsaturated fatty acids may be involved in the regulation of blood‐brain barrier (Na++ K+)‐ATPase and may play a role in the cerebral dysfunction associated with diseases in which plasma levels of nonesterified fatty acids are elevated.

Alterations of cerebromicrovascular Na+, K+-ATPase activity due to fatty acids and acute hypertension

Acute hypertension, induced in rats by intravenous injection of angiotensin II, previously has been shown to increase cerebrovascular permeability to macromolecules. The purpose of this study was to examine the effect of acute hypertension on Na+,K(+)-ATPase, the enzyme responsible for controlling ionic permeability of the cerebromicrovascular endothelium. The K(+)-dependent p-nitrophenylphosphatase activity of the cerebromicrovascular Na+,K(+)-ATPase was determined using microvessels prepared from hypertensive and normotensive rats. When compared to controls, a 70% decrease (P < 0.02) in the maximum rate (Vmax) of the Na+,K(+)-ATPase from hypertensive rats was evident with no change in the Michaelis constant (KM). In contrast, gamma-glutamyltranspeptidase, a marker enzyme for cerebral endothelial cells, was not significantly affected. Sodium arachidonate (1-100 microM) inhibited the phosphatase activity of the Na+,K(+)-ATPase in microvessels isolated from both normotensive and hypertensive rats in a dose-dependent manner. Furthermore, poly-unsaturated fatty acids (sodium linoleate and arachidonate) evoked the greatest inhibition of the enzyme, while sodium oleate and sodium palmitate inhibited the Na+,K(+)-ATPase to lesser extents. This regulation of enzyme activity by fatty acids was comparable in control and hypertensive groups. In summary, the data indicate that the cerebromicrovascular Na+,K(+)-ATPase was altered as a consequence of acute hypertension and that poly-unsaturated fatty acids can modulate this enzyme in microvessels derived from hypertensive or control rats.

Calcium and the Impairment of Contractions to Norepinephrine in Aorta Isolated from the Spontaneously Hypertensive Rat

Helical strip contractility from hypertensive (SHR) and normotensive (WKY) rat aortas assessed in the presence of varying calcium concentrations indicated that SHR strips exhibit a higher intrinsic myogenic tone and contract less to norepinephrine (NE) in a physiological calcium concentration compared to controls. Relatively higher isometric tension was developed in the SHR in low calcium (0-0. 27 mM). While control responses were blunted by LaCl3, EGTA, and nifedipine, the SHR strips were unaffected. Addition of procaine significantly enhanced SHR contractility to NE with no effect on control strips. These data suggest that abnormal cytosolic calcium provokes an increase in myogenic tone and an impaired contractile response of aortic smooth muscle cells to NE.