In vivo biodistribution of 125IPIP and internal dosimetry of 123IPIP radioiodinated agents selective to the muscarinic acetylcholinergic receptor complex.

The development of new radioiodinated ligands for imaging the muscarinic acetylcholinergic complex (mAChR) using single photon emission computed tomography (SPECT) requires the evaluation of human organ doses prior to approval for human use. Animal biodistribution and excretion data were obtained and evaluated for IPIP, a new mAChR agent. Preliminary biodistribution studies were performed on four different stereoisomers of IPIP. A biokinetic model of the Z-(S)-IPIP stereoisomer was constructed for the rat and used to estimate the internal absorbed dose in humans based on an extrapolation of the rat model. The thyroid is the critical organ for this radiopharmaceutical, with an absorbed dose estimate of 2.4 mGy/MBq for both males and females, when labeled with 123I. Even when blocked, the thyroid is still the critical organ, yet with a 90% dose reduction. The heart and brain receive the next highest doses in both males and females. Effective dose estimates for the use of pure 123I-PIP in humans are 0.16 mSv/MBq for males and 0.14 mSv/MBq for females. The biodistribution studies of the Z-(S)-IPIP stereoisomer showed the most promise as a successful agent for imaging muscarinic receptor sites in the heart and brain. IPIP also demonstrated potential as a therapeutic radiopharmaceutical for some colon carcinomas where muscarinic receptor sites are expressed in the tumor cells. These results provide preliminary data for use of IPIP in clinical studies on humans.

CNS-induced deficits of heavy particle irradiation in space: the aging connection.

Our research over the last several years has suggested that young (3 mo) rats exposed to whole-body 56Fe irradiation show neuronal signal transduction alterations and accompanying motor behavioral changes that are similar to those seen in aged (22-24 mo) rats. Since it has been postulated that 1-2% of the composition of cosmic rays contain 56Fe particles of heavy particle irradiation, there may be significant CNS effects on astronauts on long-term space flights which could produce behavioral changes that could be expressed during the mission or at some time after the return. These, when combined with other effects such as weightlessness and exposure to proton irradiations may even supercede mutagenic effects. It is suggested that by determining mechanistic relationships that might exist between aging and irradiation it may be possible to determine the common factor(s) involved in both perturbations and develop procedures to offset their deleterious effects. For example, one method that has been effective is nutritional modification.

Radiation-induced progressive decrease in fluid secretion in rat submandibular glands is related to decreased acinar volume and not impaired calcium signaling.

The mechanism(s) of radiation-induced salivary gland dysfunction is poorly understood. In the present study, we have assessed the secretory function (muscarinic agonist-stimulated saliva flow, intracellular calcium mobilization, Na+/K+/2Cl- cotransport activity) in rat submandibular glands 12 months postirradiation (single dose, 10 Gy). The morphological status of glands from control and irradiated rats was also determined. Pilocarpine-stimulated salivary flow was decreased by 67% at 12 months (but not at 3 months) after irradiation. This was associated with a 47% decrease in the wet weight of the irradiated glands. Histological and morphometric analysis demonstrated that acinar cells were smaller and occupied relatively less volume and convoluted granular tubules were smaller but occupied the same relative volume, while intercalated and striated ducts maintained their size but occupied a greater relative volume in submandibular glands from irradiated compared to control animals. In addition, no inflammation or fibrosis was observed in the irradiated tissues. Carbachol- or thapsigargin-stimulated mobilization of Ca2+ was similar in dispersed submandibular gland cells from control and irradiated animals. Further, [Ca2+]i imaging of individual ducts and acini from control and irradiated groups showed, for the first time, that mobilization of Ca2+ in either cell type was not altered by the radiation treatment. The carbachol-stimulated, bumetanide-sensitive component of the Na+/K+/ 2Cl- cotransport activity was also similar in submandibular gland cells from control and irradiated animals. These data demonstrate that a single dose of gamma radiation induces a progressive loss of submandibular gland tissue and function. This loss of salivary flow is not due to chronic inflammation or fibrosis of the gland or an alteration in the neurotransmitter signaling mechanism in the acinar or ductal cells. The radiation-induced decrease in fluid secretion appears to be related to a change in either the water-handling capacity of the acini or the number of acinar cells in the gland.

CNS effects of heavy particle irradiation in space: behavioral implications.

Research from several sources indicates that young (3 mo) rats exposed to heavy particle irradiation (56Fe irradiation) produces changes in motor behavior as well as alterations in neuronal transmission similar to those seen in aged (22-24 mo) rats. These changes are specific to neuronal systems that are affected by aging. Since 56Fe particles make up approximately 1-2% of cosmic rays, these findings suggest that the neuronal effects of heavy particle irradiation on long-term space flights may be significant, and may even supercede subsequent mutagenic effects in their mission capabilities. It is suggested that among other methods, it may be possible to utilize nutritional modification procedures to offset the putative deleterious effects of these particles in space.

Iron-56 irradiation diminishes muscarinic but not alpha 1-adrenergic-stimulated low-Km GTPase in rat brain.

Initial findings from our laboratory have indicated that muscarinic enhancement of K(+)-evoked release of dopamine from perifused striatal slices is reduced after exposure to 56Fe-particle irradiation. This finding suggested that there is a radiation-induced deficit in muscarinic receptor sensitivity. Subsequent findings have indicated that at least part of the loss in sensitivity may occur as a result of alterations in the initial steps of the signal transduction process and involve muscarinic receptor-G protein coupling/uncoupling. The present study was carried out to localize this deficit further by determining carbachol-stimulated low-Km guanosine triphosphatase (GTPase) activity in striatal and hippocampal tissue obtained from rats exposed to 0, 0.1 or 1.0 Gy of 56Fe-particle irradiation. In addition, to examine the specificity of the effect of 56Fe-particle irradiation, alpha 1-adrenergic-stimulated low-Km GTPase activity was also examined in these tissues. The results showed that there was a high degree of specificity in the effects of 56Fe particles. Decrements were observed in muscarinic-stimulated low-Km GTPase in striatum but not in hippocampus, and 56Fe-particle irradiation did not affect alpha 1-adrenergic low-Km GTPase activity in either brain tissue.

Reductions of 56Fe heavy-particle irradiation-induced deficits in striatal muscarinic receptor sensitivity by selective cross-activation/inhibition of second-messenger systems.

Recent experiments have revealed radiation-induced (600 MeV/u 56Fe energetic particles) losses of sensitivity of rodent neostriatal muscarinic receptors to stimulation by cholinergic agonists that appears as reductions in oxotremorine enhancement of K(+)-evoked dopamine release. These losses were postulated to be the result of radiation-induced alterations early in phosphoinositide-mediated signal transduction. Additional findings indicated that if the ligand-receptor-G protein interface was by passed no radiation deficits were seen. In the present study, radiation-induced deficits in K(+)-evoked dopamine release were examined in perifused striatal tissue obtained from rats exposed to 0, 0.1 or 1.0 Gy of 56Fe particles (600 MeV/u). Results showed that these deficits could be reduced by co-applying combinations of various pharmacological agents that were known to have differential effects on various second messengers such as 1,4,5-inositol-trisphosphate (IP3). Combinations included oxotremorine-carbachol, and either oxotremorine or carbachol with arginine vasopressin or arachidonic acid. These results are discussed in terms of putative radiation-induced changes in receptor-containing membranes which alter receptor-G protein coupling/uncoupling.

Deficits in the sensitivity of striatal muscarinic receptors induced by 56Fe heavy-particle irradiation: further "age-radiation" parallels.

We had previously shown that there was a loss of sensitivity of muscarinic receptors (mAChR) to stimulation by cholinergic agonists (as assessed by examining oxotremorine enhancement of K(+)-evoked release of dopamine from neostriatal slices) in animals that had been exposed to energetic particles (56Fe, 600 MeV/n), an important component of cosmic rays. This loss of mAChR sensitivity was postulated to be the result of radiation-induced alterations in phosphoinositide-mediated signal transduction. The present experiments were undertaken as a first step toward determining the locus of these radiation-induced deficits in signal transduction by examining K+ enhancement of release of dopamine in 56Fe-exposed animals (0, 0.1, and 1.0 Gy) with agents [A23187, a potent Ca2+ ionophore, or 1,4,5-inositol trisphosphate (IP3)] that "bypass" the mAChR-G protein interface and by comparing the response to oxotremorine-enhanced K(+)-evoked release of dopamine. Results showed that although oxotremorine-enhanced K(+)-evoked release of dopamine was reduced significantly in the radiation groups, no radiation effects were seen when A23187 or IP3 was used to enhance K(+)-evoked release of dopamine. Since similar findings have been observed in aging, the results are discussed in terms of the parallels between aging and radiation effects in signal transduction that might exist in the neostriatum.

Naltrexone pretreatment blocks microwave-induced changes in central cholinergic receptors.

Repeated exposure of rats to pulsed, circularly polarized microwaves (2,450-MHz, 2-microseconds pulses at 500 pps, power density 1 mW/cm2, at an averaged, whole-body SAR of 0.6 W/kg) induced biphasic changes in the concentration of muscarinic cholinergic receptors in the central nervous system. An increase in receptor concentration occurred in the hippocampus of rats subjected to ten 45-min sessions of microwave exposure, whereas a decrease in concentration was observed in the frontal cortex and hippocampus of rats exposed to ten 20-min sessions. These findings, which confirm earlier work in the authors' laboratory, were extended to include pretreatment of rats with the narcotic antagonist naltrexone (1 mg/kg, IP) before each session of exposure. The drug treatment blocked the microwave-induced changes in cholinergic receptors in the brain. These data further support the authors' hypothesis that endogenous opioids play a role in the effects of microwaves on central cholinergic systems.

Muscarinic receptor size on smooth muscle cells and membranes.

The loss of [3H]quinuclidinyl benzilate ([3H]QNB) binding following high-energy radiation was used to compare the muscarinic receptor size on single smooth muscle cells isolated by collagenase digestion from the canine stomach and on plasma membranes derived from intact gastric smooth muscle without exposure to exogenous proteolysis. Radiation inactivation of galactose oxidase (68 kdaltons), yeast alcohol dehydrogenase (160 kdaltons), and pyruvate kinase (224 kdaltons) activities were used as molecular-weight standards. Radiation inactivation of [3H]QNB binding to rat brain membranes, which gave a target size of 86 kdaltons, served as an additional control. In isolated smooth muscle cells, the calculated size of the muscarinic receptor was 80 +/- 8 kdaltons. In contrast, in a smooth muscle enriched plasma membrane preparation, muscarinic receptor size was significantly smaller at 45 +/- 3 kdaltons. Larger molecular sizes were obtained either in the presence of protease inhibitors (62 +/- 4 kdaltons) or by using a crude membrane preparation of gastric smooth muscle 86 +/- 7 kdaltons).

Serotonin-S2 and dopamine-D2 receptors are the same size in membranes.

Target size analysis was used to compare the sizes of serotonin-S2 and dopamine-D2 receptors in rat brain membranes. The sizes of these receptors were standardized by comparison with the muscarinic receptor, a receptor of known size. The number of serotonin-S2 receptors labeled with (3H)ketanserin or (3H)spiperone in frontal cortex decreased as an exponential function of radiation dose, and receptor affinity was not affected. The number of dopamine-D2 receptors labeled with (3H)spiperone in striatum also decreased as an exponential function of radiation dose, and D2 and S2 receptors were equally sensitive to radiation. In both striatum and frontal cortex, the number of muscarinic receptors labeled with (3H)QNB decreased as an exponential function of radiation dose, and were much less sensitive to radiation than S2 and D2 receptors. These data indicate that in rat brain membranes, S2 and D2 receptors are of similar size, and both molecules are much larger than the muscarinic receptor.

Molecular characterization of muscarinic receptor subtypes in bovine cerebral cortex by radiation inactivation and molecular exclusion h.p.l.c.

Muscarinic receptor subtypes in bovine cerebral cortex were investigated by means of radiation inactivation and molecular exclusion high performance liquid chromatography (h.p.l.c.). The functional molecular size of the muscarinic receptor in situ was determined by the radiation inactivation method. The value for the muscarinic receptor labelled with [3H]-quinuclidinylbenzilate ([3H]-QNB) was 91,000 daltons, while that labelled with [3H]-pirenzepine [( 3H]-PZ) was 157,000 daltons. The muscarinic receptor solubilized with digitonin could be labelled with [3H]-PZ as well as with [3H]-QNB. 3-[(3-Cholamidopropyl)-dimethylammonio] - propane sulphonate (CHAPS) solubilized the muscarinic receptor labelled with [3H]-QNB but not that labelled with [3H]-PZ, in agreement with the low affinity of pirenzepine for inhibiting [3H]-QNB binding in CHAPS-solubilized preparations. The size of the muscarinic receptor in solution was estimated by molecular exclusion h.p.l.c. The digitonin-solubilized muscarinic receptor had a molecular weight of 290,000 and the [3H]-QNB and [3H]-PZ binding activities behaved identically. The CHAPS-solubilized muscarinic receptor labelled with [3H]-QNB was apparently of high molecular weight (greater than 1,000,000 Mr), indicating the formation of aggregates and/or micelles. In the presence of digitonin this form was dissociated into a lower molecular weight species (580,000 Mr). These data indicate that the ligand binding component of the muscarinic receptor species labelled by both [3H]-QNB and [3H]-PZ exists on the same receptor protein, but that the [3H]-PZ binding component in situ is probably coupled to other components in the membrane.

Molecular mechanism of the effects of guanine nucleotide and sulfhydryl reagent on muscarinic receptors in smooth muscles studied by radiation inactivation.

The molecular sizes of the units concerned in 3-quinuclidinyl benzilate (QNB) binding and in the effects of guanine nucleotide and sulfhydryl reagent on the inhibition of QNB binding by carbachol in smooth muscle of guinea pig ileum were determined to be 76,000, 179,000 and 107,000, respectively by the radiation inactivation method. One or more subunits (GTP subunit) other than the receptor subunit in a muscarinic receptor appeared to be involved in the effect of guanine nucleotide. When guanine nucleotide was present, the receptor subunit seemed to be dissociated from the GTP subunit.

The development of the cholinergic system in rat hippocampus following postnatal x-irradiation.

Postnatal X-irradiation of the rat hippocampus results in a marked reduction in the number of the postnatally developing granular neurons in the dentate gyrus and also caused a marked increase in the specific activity of acetylcholinesterase (AChE) and choline acetyltransferase (CAT) and a slight but consistent increase in the activity per whole hippocampus of AChE. The effect of irradiation on the granular neurons and on the cholinergic enzymes was found to be dose and age dependent. Drastic increase in specific enzymatic activities is also observed in the irradiated cerebellum whose granular neurons differentiate postnatally and to a lesser extent in the cerebral cortex in which cell formation is accomplished prior to birth. Staining for AChE activity revealed enhanced staining in the molecular layer and the hilar zone of the irradiated dentate gyrus, and in the striatum lucidum of area CA3 which corresponds to the projection area of the mossy fibers. Enhanced staining in area CA1 and subiculum was noticed especially in the supra- and infrapyramidal layers. Biochemical analysis demonstrated that AChE and CAT activities were 140-180% higher in the subareas of the irradiated vs non-irradiated hippocampus. The development and distribution of the postsynaptic muscarinic receptors in the irradiated hippocampus by [3H]quinuclidinyl benzilate (QNB)-binding were also studied. It was found that the elimination of the postnatally formed neurons does not appear to change the developmental pattern of the [3H]QNB-binding sites but reduced receptor level to about 75% of control to adulthood. Measurements of the [3H]QNB-binding in the subareas within the hippocampus revealed marked reduction in the specific [3H]QNB-binding in the molecular layer of the dentate gyrus but not in other subareas. However, the reduction in [3H]QNB-binding sites in the dentate is not as drastic as the reduction in the number of granular neurons. It is suggested that muscarinic sites may be located on early formed neurons, non-cholinergic afferents, or glial elements in this area.