Expression of human muscarinic cholinergic receptors in tobacco

We expressed human m1, m2 and chimeric muscarinic cholinergic receptors (MAChR) in tobacco plants and in cultured BY2 tobacco cells using Agrobacterium-mediated transformation. The membranes of most transgenic plants and calli bound muscarinic ligands with appropriate affinities, kinetics and pharmacologic specificity, as determined by direct and competitive binding measurements using the muscarinic ligand [3H]quinuclidinyl benzylate (QNB). Membranes of untransformed plants and calli or those transformed with vector alone did not bind [3H]QNB. Preliminary experiments did not suggest regulation of endogenous plant G protein signalling pathways by the recombinant receptors. Membranes from one callus clone expressed m1 MAChR at the level of 2.0–2.5 pmol [3H]QNB bound per mg membrane protein, more than the number of m1 MAChR in mammalian brain and comparable to that expressed in Sf9 insect cells using baculovirus vectors. This work demonstrates high level expression of active G protein-coupled receptors in plants, such that signaling might be genetically reconstituted by co-expression of appropriate G proteins and effectors.     

Maturation of mammalian myocardial muscarinic cholinergic receptors

Biochemical alterations of the cardiac muscarinic binding sites have not been correlated with physiological observations. The development of responsiveness to acetylcholine in the fetal mouse heart occurs during the third trimester. We tested the hypothesis that the altered physiological response was related to changes in the muscarinic cholinergic binding site assayed by using the potent antagonist [³H]-quinuclidinyl benzilate (QNB). Analysis of saturation isotherms of specific (atropine displaceable) [³H]-QNB binding gave an apparent dissociation constant (K_Dapp) of 27 ± 3 (SEM) pM for adult heart. Receptor density increased significantly during the third trimester of pregnancy to 48% of adult, while the K_Dapp did not change. The increase in receptor density parallels the demonstrated increased responsiveness to acetylcholine.     

Local heating of human skin by millimeter waves: a kinetics study

Heating rates of human skin exposed locally to 42.25 GHz mm waves, coming from a waveguide (WG) opening or a YAV device designed for therapeutic application, were studied in vivo using infrared (IR) thermography. For both radiators, the power density distribution was described by a circularly symmetrical Gaussian type function on the exposed skin surface. Insertion of a small thermocouple (d = 0.1 mm) in the exposed area did not produce any significant artifact, either in the power density distribution or kinetics measurement, providing it was perpendicular to the E vector. The heating kinetics in the skin exposed with either the WG opening or the YAV device were well fitted to solutions of the 2-D bio-heat transfer equation for homogeneous tissue. Changes in irradiating beam size (1-8 mm) had no detectable effect on the initial (0.3-3.0 s) phase of the heating kinetics. However, the amplitude of the kinetics decreased substantially with decreasing the beam size. As the temperature rise in the time interval necessary for reliable measurement of the initial temperature rise rate was very small, an accurate experimental determination of specific absorption rate (SAR) becomes practically impossible at the low intensities normally used in our experiments. The correct SAR values may be found from fitting of the model to the heating kinetics. Bioelectromagnetics 24:571-581, 2003.     

Prejunctional inhibitory muscarinic receptors on cholinergic nerves in human and guinea pig airways

We have investigated whether prejunctional inhibitory muscarinic receptors ("autoreceptors") exist on cholinergic nerves in human airways in vitro and whether guinea pig trachea provides a good model for further pharmacological characterization of these receptors. Pilocarpine was used as a selective agonist and gallamine as a selective antagonist of these autoreceptors. Acetylcholine (ACh) release from postganglionic cholinergic nerves was elicited by electrical field stimulation (EFS) (40 V, 0.5 ms, 32 Hz). In human bronchi, pilocarpine inhibited the contractile response to EFS in a dose-related fashion; the dose inhibiting 50% of the control contraction was 2.2 +/- 0.4 x 10(-7) (SE) M (n = 22), and the inhibition was 96% at 3 x 10(-5) M. The inhibitory effects of pilocarpine were antagonized by gallamine in a dose-related fashion. The results were qualitatively the same in the guinea pig. Gallamine significantly enhanced the contractile response to EFS in the guinea pig, whereas pirenzepine failed to do so, which suggests that M2-receptors are involved. We conclude that prejunctional muscarinic receptors that inhibit ACh release are present on cholinergic nerves in human airways and that guinea pig trachea is a good model for further pharmacological characterization of these receptors, which appear to belong to the M2-subtype.     

Agonist-independent phosphorylation of purified cardiac muscarinic cholinergic receptors by protein kinase C

The results of several studies have suggested that muscarinic cholinergic receptors (mAChR) may be regulated by multiple pathways involving phosphorylation of the receptors. Previous studies have demonstrated that chick heart mAChR are phosphorylated by the beta-adrenergic receptor kinase (beta-AR kinase) in an agonist-dependent manner, and it has been suggested that this process may be linked to receptor desensitization. In this work, we present evidence that protein kinase C can phosphorylate the purified, reconstituted chick heart mAChR and can modify the interaction of the receptors with GTP binding proteins (G-proteins) that couple the receptors to effectors. Phosphorylation of the mAChR with protein kinase C occurred to an extent of approximately 5 mol of P/mol of receptor. Neither the rate nor the extent of the protein kinase C mediated phosphorylation of mAChR was agonist-dependent. Under the conditions tested, the initial rate of phosphorylation of the mAChR by protein kinase C was significantly more rapid than that obtained with the beta-AR kinase. At equilibrium, phosphorylation of mAChR by protein kinase C and beta-AR kinase was partially additive. The functional effects of protein kinase C mediated phosphorylation of the mAChR were assessed by comparing the abilities of purified G-proteins (Gi and Go) to reconstitute high-affinity agonist binding to phosphorylated and nonphosphorylated receptors. A significantly larger percentage of the receptors phosphorylated with protein kinase C exhibited G-protein-dependent high-affinity agonist binding, suggesting that phosphorylation of the receptors by protein kinase C modulates receptor function in a positive manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Local heating of human skin by millimeter waves: effect of blood flow

We investigated the influence of blood perfusion on local heating of the forearm and middle finger skin following 42.25 GHz exposure with an open ended waveguide (WG) and with a YAV mm wave therapeutic device. Both sources had bell-shaped distributions of the incident power density (IPD) with peak intensities of 208 and 55 mW/cm(2), respectively. Blood perfusion was changed in two ways: by blood flow occlusion and by externally applied vasodilator (nonivamide/nicoboxil) cream to the skin. For thermal modeling, we used the bioheat transfer equation (BHTE) and the hybrid bioheat equation (HBHE) which combines the BHTE and the scalar effective thermal conductivity equation (ETCE). Under normal conditions with the 208 mW/cm(2) exposure, the cutaneous temperature elevation (DeltaT) in the finger (2.5 +/- 0.3 degrees C) having higher blood flow was notably smaller than the cutaneous DeltaT in the forearm (4.7 +/- 0.4 degrees C). However, heating of the forearm and finger skin with blood flow occluded was the same, indicating that the thermal conductivity of tissue in the absence of blood flow at both locations was also the same. The BHTE accurately predicted local hyperthermia in the forearm only at low blood flow. The HBHE made accurate predictions at both low and high perfusion rates. The relationship between blood flow and the effective thermal conductivity (k(eff)) was found to be linear. The heat dissipating effect of higher perfusion was mostly due to an apparent increase in k(eff). It was shown that mm wave exposure could result in steady state heating of tissue layers located much deeper than the penetration depth (0.56 mm). The surface DeltaT and heat penetration into tissue increased with enlarging the irradiating beam area and with increasing exposure duration. Thus, mm waves at sufficient intensities could thermally affect thermo-sensitive structures located in the skin and underlying tissue.     

Reconstitution of solubilized atrial cholinergic muscarinic receptors in liposomes

The reconstitution of solubilized bovine atrial cholinergic muscarinic receptor into liposomes made of exogenous lipids has been achieved by polyethyleneglycol precipitation. Of the different lipid mixtures used, soybean lecithins were shown to be the best on the basis of receptor recovery. The receptor reconsituted into soybean lecithins liposomes exhibited ligand binding properties very similar to those of the native receptor. The dissociation constant of [³H]-N-methyl-scopolamine ([³H]NMS) was 0.46 and 0.30 nM as determined by equilibrium and kinetics experiments respectively. The potency of a range of muscarinic ligands in displacing [³H]NMS binding was atropine > methyl-atropine > scopolamine > pirenzepine oxotremorine > gallamine > carbamylcholine > pilocarpine bethanechol. The Hill slopes of the displacement curves were near 1 for the antagonists and smaller than 1 for the agonists and for gallamine. The agonist binding may be modulated by guanine nucleotides. These results indicate that soybean lecithins fulfill the lipid requirements for the reconstitution of the atrial muscarinic receptor.     

Phosphorylation of chick heart muscarinic cholinergic receptors by the beta-adrenergic receptor kinase

Previous studies have demonstrated that muscarinic cholinergic receptors (mAChR) become markedly phosphorylated when intact cardiac cells are stimulated with a muscarinic agonist. This process appears to be related to the process of receptor desensitization. However, the mechanism of agonist-induced phosphorylation of mAChR is not known. In situ phosphorylation studies suggested that agonist-induced phosphorylation of mAChR may involve the participation of a receptor-specific kinase and/or require agonist occupancy. These observations regarding phosphorylation and desensitization of mAChR are similar to observations made for beta-adrenergic receptors. Recent studies have indicated that homologous desensitization of beta-adrenergic receptors may be due to the phosphorylation of these receptors by a novel protein kinase that only recognizes the agonist-occupied form of the receptors. As muscarinic receptors are structurally homologous to beta-adrenergic receptors, we have initiated studies to identify the protein kinase responsible for the phosphorylation of muscarinic receptors by determining whether the chick heart muscarinic receptor would serve as a substrate for the beta-adrenergic receptor kinase (beta-AR kinase). We report that the purified and reconstituted chick heart muscarinic receptor serves as an excellent substrate in vitro for the beta-AR kinase. Phosphorylation of mAChR receptors by the beta-AR kinase was only observed in the presence of a muscarinic receptor agonist and was prevented in the presence of antagonist. Both the extent of phosphorylation (3-4 mol of P/mol of receptor) and the phosphoamino acid composition of the mAChR after incubation in vitro with beta-AR kinase were similar to the characteristics of agonist-induced phosphorylation of mAChR in situ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cooperativity and oligomeric status of cardiac muscarinic cholinergic receptors

Muscarinic cholinergic receptors can appear to be more numerous when labeled by [(3)H]quinuclidinylbenzilate (QNB) than by N-[(3)H]methylscopolamine (NMS). The nature of the implied heterogeneity has been studied with M(2) receptors in detergent-solubilized extracts of porcine atria. The relative capacity for [(3)H]NMS and [(3)H]QNB was about 1 in digitonin-cholate, 0.56 in cholate-NaCl, and 0.44 in Lubrol-PX. Adding digitonin to extracts in cholate-NaCl increased the absolute capacity for both radioligands, and the relative capacity increased to near 1. The latency cannot be attributed to a chemically impure radioligand, instability of the receptor, an irreversible effect of NMS, or a failure to reach equilibrium. Binding at near-saturating concentrations of [(3)H]QNB in cholate-NaCl or Lubrol-PX was blocked fully by unlabeled NMS, which therefore appeared to inhibit noncompetitively at sites inaccessible to radiolabeled NMS. Such an effect is inconsistent with the notion of functionally distinct, noninterconverting, and mutually independent sites. Both the noncompetitive effect of NMS on [(3)H]QNB and the shortfall in capacity for [(3)H]NMS can be described quantitatively in terms of cooperative interactions within a receptor that is at least tetravalent; no comparable agreement is possible with a receptor that is only di- or trivalent. The M(2) muscarinic receptor therefore appears to comprise at least four interacting sites, presumably within a tetramer or larger array, and ligands appear to bind in a cooperative manner under at least some conditions.     

Agonist-induced phosphorylation and desensitization of human m2 muscarinic cholinergic receptors in Sf9 insect cells.

The human m1 (hm1) and m2 (hm2) muscarinic cholinergic receptors (mAChR) expressed in Sf9 insect cells using recombinant baculovirus were tested for their ability to undergo agonist-dependent phosphorylation and desensitization. The muscarinic agonist carbachol induced phosphorylation of the hm2 mAChR in the Sf9 cells incubated with 32P(i) to an extent of 4-5 mol of phosphate/mol of receptor. In contrast, no phosphorylation of the hm1 mAChR was observed. The hm2 mAChR stimulated [35S]GTP gamma S binding to, and GTPase activity of, the insect cell G-proteins. These receptor-mediated activities were reduced by 50% in membranes prepared from agonist-treated cells compared to control, suggesting that the agonist-induced phosphorylation of the hm2 mAChR resulted in desensitization of the receptors. No role for protein kinase C or cyclic nucleotide-dependent kinases in receptor phosphorylation and desensitization was suggested from studies using agents known to modulate the activity of these enzymes. However, pertussis toxin was found to completely eliminate the interaction of the hm2 receptors with the insect cell G-proteins, but did not perturb the ability of carbachol to induce agonist-dependent phosphorylation of the receptors. These results suggested that G-proteins and/or G-protein-activated signalling were not necessary for the agonist-induced phosphorylation of the receptors. Overall, the data indicated that the human m2 (but not the human m1) mAChR expressed in Sf9 insect cells undergo phosphorylation and desensitization in an agonist-dependent, G-protein-independent fashion by an endogenous insect cell kinase. The results demonstrated that a human G-protein-linked receptor is regulated in insect cells in a manner that is similar to that involving members of the G-protein receptor-kinase family.     

beta-Adrenergic and muscarinic cholinergic receptors in rat submaxillary glands. Effects of thyroidectomy.

Administration of triiodothyronine to thyroidectomized rats increased the density of beta-adrenergic receptors in rat submaxillary gland without significantly changing the density of muscarinic cholinergic receptors. Thus, thyroid hormone appears to regulate beta-adrenergic sensitivity in the rat salivary gland.     

Agonist-induced alteration in the membrane form of muscarinic cholinergic receptors

Incubation of 1321N1 human astrocytoma cells with carbachol resulted in a rapid loss of binding of [3H]N-methylscopolamine ([3H]NMS) to muscarinic cholinergic receptors measured at 4 degrees C on intact cells; loss of muscarinic receptors in lysates from the same cells measured with [3H]quinuclidinyl benzilate [( 3H]QNB) at 37 degrees C occurred at a slower rate. Upon removal of agonist from the medium, the lost [3H]NMS binding sites measured on intact cells recovered with a t1/2 of approximately 20 min, but only to the level to which [3H]QNB binding sites had been lost; no recovery of "lost" [3H]QNB binding sites occurred over the same period. Based on these data and the arguments of Galper et al. (Galper, J. B., Dziekan, L. C., O'Hara, D. S., and Smith, T. W. (1982) J. Biol. Chem. 257, 10344-10356) regarding the relative hydrophilicity of [3H]NMS versus [3H]QNB, it is proposed that carbachol induces a rapid sequestration of muscarinic receptors that is followed by a loss of these receptors from the cell. These carbachol-induced changes are accompanied by a change in the membrane form of the muscarinic receptor. Although essentially all of the muscarinic receptors from control cells co-purified with the plasma membrane fraction on sucrose density gradients, 20-35% of the muscarinic receptors from cells treated for 30 min with 100 microM carbachol migrated to a much lower sucrose density. This conversion of muscarinic receptors to a "light vesicle" form occurred with a t1/2 approximately 10 min, and reversed with a t1/2 approximately 20 min. In contrast to previous results in this cell line regarding beta-adrenergic receptors (Harden, T. K., Cotton, C. U., Waldo, G. L., Lutton, J. K., and Perkins, J. P. (1980) Science 210, 441-443), agonist binding to muscarinic receptors in the light vesicle fraction obtained from carbachol-treated cells was still regulated by GTP. One interpretation of these data is that agonists induce an internalization of muscarinic receptors with the retention of their functional interaction with a guanine nucleotide regulatory protein.     

Increased muscarinic cholinergic receptors in prefrontal cortices of medicated schizophrenics

Alterations in 3H-quinuclidinyl benzilate binding sites associated with muscarinic cholinergic receptors were investigated in orbito-frontal and medial frontal cortices from 12 schizophrenics, 6 on-drug and 6 off-drug cases, and from 10 controls. Significantly lower affinities of the sites were found in both areas of schizophrenics than controls. An increase in receptor number was shown only in the orbito-frontal cortex from schizophrenics. On-drug group of schizophrenics did, however, show a significant increase in receptor number and a significant decrease in affinity in both areas, while there were no significant differences in any binding parameters of off-drug schizophrenics from controls. Also in the caudate the similar results were obtained. It is, thus, concluded that alterations in muscarinic cholinergic receptors of schizophrenic patients result from long-term medication with antimuscarinic actions.     

Reconstitution of resolved muscarinic cholinergic receptors with purified GTP-binding proteins

The association of agonists with muscarinic receptors in membranes from bovine brain was affected only slightly by guanine nucleotides. However, solubilization of these membranes with deoxycholate and subsequent removal of detergent resulted in a preparation of receptors with increased affinity for agonists and a large increase in response to guanine nucleotides. Chromatography of deoxycholate extracts of membranes on DEAE-Sephacel resulted in the separation of receptors from 95% of the guanine nucleotide-binding activity. Guanine nucleotides had no effect on the binding of agonists to these resolved receptors. The effect of guanine nucleotides was restored after the addition of either of two purified guanine nucleotide-binding proteins from bovine brain. One of these proteins, presumably brain GI, is composed of subunits with the same molecular weights (alpha, 41,000; beta, 35,000; gamma, 11,000) and functions as the inhibitory guanine nucleotide-binding protein isolated from liver. The other protein, termed Go, is a novel guanine nucleotide-binding protein that possesses a similar subunit composition (alpha, 39,000; beta, 35,000; gamma, 11,000) but whose function is not yet known. Addition of either protein to the resolved receptor preparation increased agonist affinity by at least 10-20-fold, and low concentrations of guanine nucleotides specifically reversed this effect. Reconstitution of receptors with the resolved subunits of Go demonstrates that the beta subunit alone had no effect on agonist binding, but that this subunit does appear to enhance the effects observed with the alpha subunit alone.     

The non-neuronal cholinergic system of human skin.

In human skin both resident and transiently residing cells are part of the extra- or non-neuronal cholinergic system, creating a highly complex and interconnected cosmos in which acetylcholine (ACh) and choline are the natural ligands of nicotinic and muscarinic receptors with regulatory function in both physiology and pathophysiology. ACh is produced in keratinocytes, endothelial cells and most notably in immune competent cells invading the skin at sites of inflammation. The cholinergic system is involved in basic functions of the skin through autocrine, paracrine, and endocrine mechanisms, like keratinocyte proliferation, differentiation, adhesion and migration, epidermal barrier formation, pigment-, sweat- and sebum production, blood circulation, angiogenesis, and a variety of immune reactions. The pathophysiological consequences of this complex cholinergic "concert" are only beginning to be understood. The present review aims at providing insight into basic mechanisms of this highly complex system.     

Activation of MAP kinase by muscarinic cholinergic receptors induces cell proliferation and protein synthesis in human breast cancer cells

Carbachol (Cch), a muscarinic acetylcholine receptor (mAChR) agonist, increases intracellular-free Ca(2+) mobilization and induces mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) phosphorylation in MCF-7 human breast cancer cells. Pretreatment of cells with the selective phospholipase C (PLC) inhibitor U73122, or incubation of cells in a Ca(2+)-free medium did not alter Cch-stimulated MAPK/ERK phosphorylation. Phosphorylation of MAPK/ERK was mimicked by phorbol 12-myristate acetate (PMA), an activator of protein kinase C (PKC), but Cch-evoked MAPK/ERK activation was unaffected by down-regulation of PKC or by pretreatment of cells with GF109203X, a PKC inhibitor. However, Cch-stimulated MAPK/ERK phosphorylation was completely blocked by myristoylated PKC-zeta pseudosubstrate, a specific inhibitor of PKC-zeta, and high doses of staurosporine. Pretreatment of human breast cancer cells with wortmannin or LY294002, selective inhibitors of phosphoinositide 3-kinase (PI3K), diminished Cch-mediated MAPK/ERK phosphorylation. Similar results were observed when MCF-7 cells were pretreated with genistein, a non-selective inhibitor of tyrosine kinases, or with the specific Src tyrosine kinase inhibitor PP2. Moreover, in MCF-7 human breast cancer cells mAChR stimulation induced an increase of protein synthesis and cell proliferation, and these effects were prevented by PD098059, a specific inhibitor of the mitogen activated kinase kinase. In conclusion, analyses of mAChR downstream effectors reveal that PKC-zeta, PI3K, and Src family of tyrosine kinases, but not intracellular-free Ca(2+) mobilization or conventional and novel PKC activation, are key molecules in the signal cascade leading to MAPK/ERK activation. In addition, MAPK/ERK are involved in the regulation of growth and proliferation of MCF-7 human breast cancer cells.     

Functional effects of protein kinase C-mediated phosphorylation of chick heart muscarinic cholinergic receptors.

Muscarinic cholinergic receptors (mAChR) purified from chick heart were phosphorylated by protein kinase C (PKC) and reconstituted with the purified GTP-binding regulatory protein Go. The effects of PKC phosphorylation on the interaction of mAChR with Go were assessed by monitoring for agonist-stimulated guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) binding to Go, agonist-stimulated GTPase activity of Go, and the capability of Go to induce high affinity agonist binding to mAChR. Both the receptor-stimulated GTP gamma S binding and GTPase activity of Go were markedly diminished as a result of PKC-mediated phosphorylation of the mAChR, whereas the ability of Go to induce high affinity agonist binding to the receptors was unaffected. When mAChR were first reconstituted with Go and then subjected to phosphorylation with PKC, a complete inhibition of the phosphorylation of mAChR by PKC was observed. The inhibitory effect of Go on mAChR phosphorylation was concentration-dependent and was prevented by the presence of GTP gamma S in the reaction mixtures. Taken together, these results indicate that the phosphorylation of mAChR by PKC modulates receptor/G-protein interactions and that the ability of the receptors to act as substrates for PKC may be regulated by receptor/G-protein interactions.     

Transport of muscarinic cholinergic receptors in the sciatic nerve of rat

On the basis of the specific [³H]quinuclidinyl-benzilate binding, the transport of muscarinic cholinergic receptors has been demonstrated in the ventral horn, sciatic nerve and in the 3 mm segments proximal and distal to the ligature of rat sciatic nerves ligated for 24 h (a) without electrolytic lesion, (b) six days after lesion of the spinal ganglia, (c) six days after lesion of the motoric axons, and (d) six days after transection of the sciatic nerve. The distribution of these receptors was also studied in the ventral spinal horn, dorsal root sensory axons, spinal ganglia and sciatic nerve of rabbit.Our results suggest that the receptors are transported in the sciatic nerve of rat. This transport consists of a large anterograde, and a discrete retrograde flow of muscarinic cholinergic receptors. Most of the receptors are possibly synthesized in the motoneuron cell bodies and migrate in the motoric axons; to a lesser extent they may also be synthesized in the cell bodies of the dorsal root ganglia and migrate in the sensory axons of the sciatic nerve.