Using pharmacy technicians and electronic health record capabilities to improve outcomes for patients with cardiovascular disease.

Objective: This study aimed to compare lipid and blood pressure (BP) control before and after implementing a certified pharmacy technician (CPhT) protocol that optimized electronic health record (EHR) capabilities and shifted work from clinical pharmacy specialists (CPSs) to CPhT.

Setting: Kaiser Permanente Colorado's pharmacist-managed cardiac risk reduction service (which manages dyslipidemia, hypertension, and diabetes for all patients with atherosclerotic cardiovascular disease).

Practice Description: In 2019, a protocol that optimized EHR capabilities and allowed work to be offloaded from CPS to CPhT was implemented.

Structure/function studies on the activation of the rainbow trout melanocortin-2 receptor.

Functional expression of the rainbow trout (rt) melanocortin-2 receptor (MC2R) in CHO cells requires co-expression with a teleost melanocortin-2 receptor accessory protein (MRAP) such as zebrafish (zf) MRAP. Transiently transfected rtMC2R/zfMRAP1 CHO cells were used to evaluate the efficacy of alanine substituted analogs of hACTH(1-24) in three motifs in the ligand: H(6)F(7)R(8)W(9), G(10)K(11)P(12)V(13)G(14), and K(15)K(16)R(17)R(18)P(19). Alanine substitution at all positions in each motif either completely blocked activation of the receptor (H(6)F(7)R(8)W(9) and K(15)K(16)R(17)R(18)P(19)) or resulted in just over 400 fold increase in EC50 value (G(10)K(11)P(12)V(13)G(14)).

Evolution of the melanocortin-2 receptor in tetrapods: studies on Xenopus tropicalis MC2R and Anolis carolinensis MC2R.

The tetrapods are a diverse assemblage of vertebrates, and that diversity is reflected in the sequences of tetrapod melanocortin-2 receptors (MC2Rs). In this review, the features common to human (mammal), Gallus gallus (bird), Anolis carolinensis (reptile), and Xenopus tropicalis (amphibian) MC2Rs in terms of ligand selectivity, requirements for interaction with MRAP1, and the effects of alanine substitutions to three amino acid motifs in the ligand hACTH(1-24) are discussed. Analysis of the effects of alanine substitutions to the H(6)F(7)R(8)W(9) and the K(15)K(16)R(17)R(18)P(19) motifs of hACTH(1-24) indicated that activation of A.

Using the human melanocortin-2 receptor as a model for analyzing hormone/receptor interactions between a mammalian MC2 receptor and ACTH(1-24).

When considering the interactions between the melanocortin peptides (i.e., ACTH, α-MSH, β-MSH, γ-MSH) and the melanocortin receptors (i.

Evolution of melanocortin receptors in cartilaginous fish: melanocortin receptors and the stress axis in elasmobranches.

There is general agreement that the presence of five melanocortin receptor genes in tetrapods is the result of two genome duplications that occurred prior to the emergence of the gnathostomes, and at least one local gene duplication that occurred early in the radiation of the ancestral gnathostomes. Hence, it is assumed that representatives from the extant classes of gnathostomes (i.e.

Identification of an MRAP-independent melanocortin-2 receptor: functional expression of the cartilaginous fish, Callorhinchus milii, melanocortin-2 receptor in CHO cells.

Phylogenetic analyses indicate that the genome of the cartilaginous fish, Callorhynchus milii (elephant shark), encodes a melanocortin-2 receptor (MC2R) ortholog. Expression of the elephant shark mc2r cDNA in Chinese hamster ovary (CHO) cells revealed that trafficking to the plasma membrane and functional activation of the receptor do not require coexpression with an exogenous melanocortin receptor-2 accessory protein (mrap) cDNA. Ligand selectivity studies indicated that elephant shark MC2R-transfected CHO cells produced cAMP in a dose-dependent manner when stimulated with either human ACTH (1-24) or [Nle(4), d-Phe(7)]-MSH.

Functional expression of Squalus acanthias melanocortin-5 receptor in CHO cells: ligand selectivity and interaction with MRAP.

The melanocortin-5 receptor (MC(5)) of the dogfish Squalus acanthias (SacMC(5) receptor) can be functionally expressed in CHO cells in the absence of the co-expression of an exogenous MRAP cDNA. Both human ACTH(1-24) and dogfish ACTH(1-25) were much better stimulators of the SacMC(5) receptor than any of the mammalian or dogfish MSH ligands that were tested. The order of ligand selectivity for the dogfish melanocortins was ACTH(1-25)>αMSH>γ-MSH=δ-MSH>β-MSH.

Functional expression of frog and rainbow trout melanocortin 2 receptors using heterologous MRAP1s.

Analysis of the functional expression of the melanocortin 2 receptor (MC2R) from a rather broad spectrum of vertebrates indicates that MC2R is exclusively selective for the ligand, ACTH, and the melanocortin receptor accessory protein 1 (MRAP1) is required for high affinity ACTH binding and activation of MC2R. A phylogenetic analysis of MRAP1 suggested that tetrapod sequences and bony fish sequences may represent two distinct trends in the evolution of the mrap1 gene. To test this hypothesis, a frog (Xenopus tropicalis) MC2R was expressed in CHO cells either in the presence of a tetrapod (mouse) MRAP1 or a bony fish (zebrafish) MRAP1.

Observations on the ligand selectivity of the melanocortin 2 receptor.

The melanocortin 2 receptor (MC2R) is unique in terms of ligand selectivity and in vitro expression in mammalian cell lines as compared to the other four mammalian MCRs. It is well established that ACTH is the only melanocortin ligand that can activate the ACTH receptor (i.e.

α-TC1.9 cells--a model system for analyzing the endoproteolytic processing of POMC.

The mouse α-TC1.9 endocrine cell line was used to analyze the amino acid requirements for endoproteolytic processing at the paired basic amino acid cleavage site, K(141)R(142) that is N-terminal to the ACTH sequence in the POMC proprotein of the anuran amphibian, Silurana tropicalis. Real-Time PCR analysis of non-transfected α-TC1.

IGF-1 receptors in Xenopus laevis ovarian follicle cells support the oocyte maturation response.

Insulin-like growth factor 1 (IGF-1)-stimulated amphibian oocyte maturation has been studied extensively by a number of laboratory groups, but in previous studies possible effects of IGF-1 on ovarian follicle cells had not been tested directly. In the study reported here, biochemical and immunofluorescent techniques were used to test Xenopus ovarian follicle cells for the presence of hormone-sensitive IGF-1 receptor. Anti-xIGF-1 receptor beta-subunit antibodies detected a 90- and 98-kDa protein doublet in manually dissected oocyte cortices (plasma membrane-vitelline envelope complexes) by protein immunoblotting both before and after removal of follicle cells from oocytes by sandpaper rolling.

Modeling the evolution of the MC2R and MC5R genes: studies on the cartilaginous fish, Heterondotus francisci.

Comparative studies support the hypothesis that the proliferation of melanocortin receptor genes (MCRs) in gnathostomes corresponds to the 2R hypothesis for the radiation of gene families in Phylum Chordata. This mini-review will initially focus on the distribution of MCRs in cartilaginous fish and the relationship between the shark MC5R gene and the proposed ancestral MC5R/2R gene. This section will be followed by the results of recent studies on the features of the ligand binding site common to all melanocortin receptors.

Recycling of the dense-core vesicle membrane protein phogrin in Min6 beta-cells.

Phogrin (IA2-beta) is an integral membrane protein of dense-core vesicles in neuroendocrine cells. We have examined the recycling of endogenous phogrin following exocytosis in insulin secreting Min6 beta-cells by monitoring stimulus dependent-uptake of antibodies directed against the lumenal domain of the protein. While low levels of internalized phogrin accumulated in LAMP1-positive lysosomes, more than 35% of internalized phogrin recycled back to an insulin-positive compartment and could return to the cell surface during a second exocytic stimulation.

Retention of peptide hormones during partial secretion in pituitary somatotrophs and corticotrophs.

The secretion of peptide hormones during exocytosis of an individual vesicle can result in either complete discharge of vesicle content or can occur in a partial manner in which some hormone is retained during transient fusion. In anterior pituitary lactotrophs, the retained hormone prolactin was internalized and recycled into a pool of vesicles that underwent preferential use during subsequent exocytic stimulations [Bauer et al., (2004) J Cell Sci.

Recycling of intact dense core vesicles in neurites of NGF-treated PC12 cells.

Exocytic fusion in neuroendocrine cells does not always result in complete release of the peptide contents from dense core vesicles (DCVs). In this study, we use fluorescence imaging and immunoelectron microscopy to examine the retention, endocytosis and recycling of chromogranin B in DCVs of NGF-treated PC12 cells. Our results indicate that DCVs retained and retrieved an intact core that was available for subsequent exocytic release.

Using FM1-43 to study neuropeptide granule dynamics and exocytosis.

In the study of neuropeptide secretion and membrane trafficking, the fluorescent dye FM1-43 provides the ability to label selectively those structures that are undergoing exocytosis and endocytosis in living cells in real time. This review describes the unique properties of the FM dyes that make them ideal for studying neuropeptide granule dynamics and discusses various techniques that take advantage of FM dyes.

Dense core vesicle dynamics in Caenorhabditis elegans neurons and the role of kinesin UNC-104.

We have developed a model system in Caenorhabditis elegans to perform genetic and molecular analysis of peptidergic neurotransmission using green fluorescent protein (GFP)-tagged IDA-1. IDA-1 represents the nematode ortholog of the transmembrane proteins ICA512 and phogrin that are localized to dense core secretory vesicles (DCVs) of mammalian neuroendocrine tissues. IDA-1::GFP was expressed in a small subset of neurons and present in both axonal and dendritic extensions, where it was localized to small mobile vesicular elements that at the ultrastructural level corresponded to 50 nm electron-dense objects in the neuronal processes.

Retention and stimulus-dependent recycling of dense core vesicle content in neuroendocrine cells.

We have used fluorescence imaging of individual exocytic events in combination with immunogold electron microscopy and FM1-43 photoconversion to study the stimulus-dependent recycling of dense core vesicle content in isolated rat pituitary lactotrophs. Secretory stimulation with high external [K(+)] resulted in 100 exocytic sites per cell that were labeled by extracellular antibodies against the peptide hormone prolactin. Morphological analysis demonstrated that the prolactin was retained and internalized in intact dense cores.

Liposomes from novel photolabile phospholipids: light-induced unloading of small molecules as monitored by PFG NMR.

A molecular dithiane-based approach to synthesis of novel photolabile phospholipids is developed. These lipids are used in formulations with egg-POPC and cholesterol to prepare light-sensitive liposomes. Irradiation of such liposomes in PBS buffer (medium pressure mercury lamp, Pyrex filter, lambda > 300 nm) significantly increases the bilayer permeability and accelerates the release of entrapped small organic molecules by an order of magnitude.

Sustained stimulation of exocytosis triggers continuous membrane retrieval in rat pituitary somatotrophs.

We studied the relationship between exocytosis and endocytosis in rat pituitary somatotrophs using patch-clamp capacitance, FM1-43 fluorescence imaging and amperometry. Stimulation of exocytosis through voltage-dependent Ca2+ channels by depolarizations (1-5 s) increased the capacitance by 4.3 +/- 0.

Intraterminal Ca(2+) and spontaneous transmitter release at the frog neuromuscular junction.

We investigated the relationship between intraterminal Ca(2+) concentration ([Ca(2+)](i)) and the frequency of miniature end plate potentials (MEPPs) at the frog neuromuscular junction by use of ratiometric imaging of fura-2-loaded nerve terminals and intracellular recording of MEPPs. Elevation of extracellular [KCl] over the range of 2-20 mM resulted in increases in [Ca(2+)](i) and MEPP frequency. Loading terminals with the fast and slow Ca(2+)-buffers bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl (BAPTA-AM) and EGTA-AM resulted in equivalent reductions in the KCl-dependent increases in MEPP frequency.

Differential regulation of granule-to-granule and granule-to-plasma membrane fusion during secretion from rat pituitary lactotrophs.

We used fluorescence imaging of individual exocytic events together with electron microscopy to study the regulation of dense core granule-to-plasma membrane fusion and granule-to-granule fusion events that occur during secretion from rat pituitary lactotrophs. Stimulating secretion with elevated extracellular potassium, with the calcium ionophore ionomycin, or with thyrotropin releasing hormone or vasoactive intestinal polypeptide resulted in abundant exocytic structures. Approximately 67% of these structures consisted of multiple granules fused together sharing a single exocytic opening with the plasma membrane, i.

Regulation of dense core release from neuroendocrine cells revealed by imaging single exocytic events.

Using FM1-43 fluorescence, we have optically detected single exocytic and endocytic events in rat pituitary lactotrophs. About fifty discrete fluorescent spots abruptly appear around the entire surface of a cell bathed in FM1-43 and high-potassium saline. The spots, which also immunostain for prolactin, reflect the labeling of dense cores as well as membranes of exocytosed secretory granules.

Monitoring secretory membrane with FM1-43 fluorescence.

FM1-43 and similar styryl dyes have proven useful as probes for membrane trafficking because they reversibly stain membranes, are impermeable to membranes, and are more fluorescent when bound to membranes than when in solution. Because these dyes stain membranes in an activity-dependent manner, they are ideal for studies of neurotransmitter release mechanisms such as synaptic vesicle recycling, exocytosis, and endocytosis. FM dyes have been used in conjunction with other techniques such as fluorescent calcium indicator dyes and electrophysiological techniques to elucidate mechanisms of presynaptic calcium homeostasis and modulation of neurotransmitter release.

The synaptic vesicle cycle.

The ins and outs of the synaptic vesicle cycle are being examined in increasing detail with diverse investigative tools in a variety of cell types, particularly those with large granules. The cycle begins with the opening of a fusion pore that connects the vesicle lumen to the extracellular fluid. Sensitive electrophysiological techniques reveal the often-stuttering behavior of single pores in non-neuronal cells, through which small molecules trickle until the fusion pore expands and the remaining contents erupt from the vesicle.