PK14105

Identification of a 17-kDa protein associated with the peripheral-type benzodiazepine receptor in vascular and other smooth muscle types

The existence of the peripheral-type benzodiazepine receptor (PBR) in vascular smooth muscle has been demonstrated in this laboratory. The present study utilized the photoaffinity ligand [3H]PK14105 to identify the protein subunit in rat aortic and other smooth muscle types to which high affinity ligands for the PBR bind. [3H]PK14105 bound to mitochondrial fractions isolated from rat aortic smooth muscle with high affinity (Kd = 7.0 +/- 0.5 nM) and high density (Bmax = 10.1 +/- 1.5 pmol/mg protein). The rank order of potency of a series of PBR ligands displacing the binding was PK11195 approximately equal to Ro5-4864 greater than protoporphyrin IX greater than flunitrazepam greater than diazepam much greater than clonazepam. [3H]PK14105 bound with comparable affinity and density to mitochondria isolated from rat myometrium and gastric smooth muscle as well. With ultraviolet irradiation, [3H]PK14105 specifically labeled a single protein of approximately 17 kDa in all three smooth muscle types examined. This protein was identical in size to that identified by [3H]PK14105 in rat adrenal gland. In adrenal gland an additional, minor protein of approximately 43 kDa was also specifically labeled by [3H]PK14105. Utilizing a probe designed from the known nucleotide sequence of the PBR in rat adrenal gland, an mRNA transcript of approximately 0.8 kilobases in size was identified in rat aortic smooth muscle by Northern blot analysis. These data indicate that a protein subunit of approximately 17 kDa comprises, at least in part, the PBR not only in vascular smooth muscle, but also in other smooth muscle types and adrenal gland as well.

Pharmacological characterization of an 18-kDa protein associated with the peripheral-type benzodiazepine receptor in salivary glands

Pharmacological characterization of peripheral type benzodiazepine receptors in rat, rabbit, mouse and human salivary glands was determined by receptor binding and photoaffinity labeling analysis using [3H]PK14105 (1-(2-fluoro-5-nitrophenyl)-3-isoquinolinecarboxylic acid). [3H]PK14105 bound to the membranes of salivary glands in rats, rabbits, mice and humans with high affinity at the nanomolar level. The rank order of receptor density in submandibular glands among several species was as follows: human > or = rat > or = mouse > rabbit. Competitive potency of receptor ligands against [3H]PK14105 was as follows: PK1195 > or = Ro5-4864 > diazepam > clonazepam > Ro15-1788. The rank order of potency against calcium channel ligands and co-transport inhibitors was as follows: nitrendipine > BAY K 8644 > bumetanide > furosemide. Pretreatment with nitrendipine or BAY K 8644 decreased the affinity of [3H]PK14105 binding to rat parotid gland membranes, without changing the density. The photoaffinity labeling with [3H]PK14105 indicated the presence of the 18-kDa protein in all salivary glands of our experiment. The inhibition of photolabeling by some receptor ligands was the same results as the receptor binding assay. In conclusion, the peripheral type benzodiazepine receptors include the 18-kDa protein photolabeled with [3H]PK14105 in salivary glands of rat, mouse, rabbit and human.

In vivo binding to peripheral benzodiazepine binding sites in lesioned rat brain: comparison between [3H]PK11195 and [18F]PK14105 as markers for neuronal damage

Peripheral-type benzodiazepine binding sites are not normally present in most cerebral tissues, but following neuronal damage, the cells involved in the ensuing gliosis show a marked expression of these sites. In a unilateral excitotoxic striatal lesion in the rat, we sought to determine whether the isoquinoline derivatives PK11195 and PK14105 bind to these sites in vivo and whether demonstration of these sites offers the potential of indirectly localising areas of neuronal damage. Binding was studied at several intervals after coinjection of [3H]PK11195 and [18F]PK14105 to determine the time courses of specific binding. Both compounds were rapidly extracted into all cerebral tissues, but in the absence of binding sites in nonlesioned tissues, this was followed by a rapid clearance of radioactivity. In lesioned areas, both [3H]PK11195 and [18F]PK14105 accumulated over the first 5 min followed by a much slower clearance of radioactivity, resulting in a "specific signal." [3H]PK11195 binding peaked at 20-30 min postinjection, with radioactivity in the lesioned striatum being three times greater than in its contralateral homologue. The specific signal was present for at least 60 min. The maximal [18 F]PK14105-specific signal was of similar magnitude but peaked earlier and was retained for only 45 min. Specific signals with both ligands were also detected in regions remote from the primary lesion site, e.g., in the hippocampus and substantia nigra. Predosing animals with a large dose of PK11195 (3 mg/kg), sufficient to saturate peripheral-type benzodiazepine binding sites, abolished in vivo binding of both [3H]PK11195 and [18F]PK14105 to both primary- and remote-lesioned tissues. The specific signal with both ligands could be of sufficient magnitude and duration to make tomographic studies in humans feasible.

Affinity labeling of membrane receptors using tissue-penetrating radiations

Photoaffinity labeling, a useful in vivo biochemical tool, is limited when applied in vivo because of the poor tissue penetration by ultraviolet (UV) photons. This study investigates affinity labeling using tissue-penetrating radiation to overcome the tissue attenuation and irreversibly label membrane receptor proteins. Using X-ray (115 kVp) at low doses (<50 cGy or Rad), specific and irreversible binding was found on striatal dopamine transporters with 3 photoaffinity ligands for dopamine transporters, to different extents. Upon X-ray exposure (115 kVp), RTI-38 and RTI-78 ligands showed irreversible and specific binding to the dopamine transporter similar to those seen with UV exposure under other conditions. Similarly, gamma rays at higher energy (662 keV) also affect irreversible binding of photoreactive ligands to peripheral benzodiazepine receptors (by PK14105) and to the dopamine (D2) membrane receptors (by azidoclebopride), respectively. This study reports that X-ray and gamma rays induced affinity labeling of membrane receptors in a manner similar to UV with photoreactive ligands of the dopamine transporter, D2 dopamine receptor (D2R), and peripheral benzodiazepine receptor (PBDZR). It may provide specific noninvasive irreversible block or stimulation of a receptor using tissue-penetrating radiation targeting selected anatomic sites.

The mitochondrial benzodiazepine receptor: evidence for association with the voltage-dependent anion channel (VDAC)

Specific, high-affinity receptors for numerous drugs have recently been localized to mitochondrial membrane proteins. This review discusses the association of the mitochondrial receptor for benzodiazepines (mBzR) with the voltage-dependent anion channel (VDAC), indicating a possible auxiliary role for VDAC as a putative drug binding protein. The proposed subunit composition of the purified mBzR complex isolated from rat kidney mitochondria includes VDAC, which functions as a recognition site for benzodiazepines (e.g., flunitrazepam), the adenine nucleotide carrier (ADC), and an 18 kDa outer membrane protein identified by covalent labelling with the mBzR antagonists isoquinoline carboxamides (e.g., PK14105).